JP2021505604A - Regulation and regulation of the function of genetically modified chimeric antigen receptor T cells with dasatinib and other tyrosine kinase inhibitors - Google Patents

Abstract

The present invention relates to the immunomodulatory characteristics of dasatinib and other tyrosine kinase inhibitors against genetically modified immune cells. The present invention is an immune cell enhancer as well as an immune cell inhibitor, depending on the administration and schedule of treatments that can be used for immunotherapy, the route of administration, sensitive receptor variants and the treatable cell types. , Dasatinib and other tyrosine kinase inhibitors.

Description

The present invention relates to the use of dasatinib and other tyrosine kinase inhibitors to control and regulate the function of genetically modified chimeric antigen receptor (CAR) -T cells in cancer immunotherapy. The present invention uses dasatinib and other tyrosine kinase inhibitors to enhance safety through the prevention and treatment of potentially life-threatening side effects that may occur during CAR-T cell immunotherapy, as well as CAR-T cells. Includes the use of dasatinib to increase the anticancer efficacy and efficacy of immunotherapy.

Adoptive immunotherapy with T cells engineered by transient or stable gene transfer to express chimeric antigen receptor (CAR) is refractory to chemotherapy and radiation therapy in hematology and oncology. Preclinical and clinical studies are being conducted as highly innovative and highly effective new treatments for malignant tumors.

CAR is a synthetic designer receptor, an extracellular antigen-binding moiety that binds to a surface molecule or structure on a tumor cell; a spacer and transmembrane domain that anchors the receptor on the surface of a T cell; and each target molecule. Or an intracellular signaling module for activating and stimulating CAR-T cells after structural binding, most commonly a co-stimulating moiety derived from CD28 or 4-1BB and the CD3 zeta domain of cis. Included in. In addition, alternative CAR designs have been developed that include the NKG2D domain, T cell receptor constant domain, and other CD3 subunits. Currently, the process of antigen binding, signaling and transduction of CAR, and subsequent T cell activation and stimulation is incomplete, at least in part, in the domain in which CAR is present in endogenous T cells (eg,). , CD3 Zetas, signaling domains such as CD28 and 4-1BB), but due to the fact that they are assembled into CAR constructs in new artificial ways.

A clinical proof of the effectiveness of CAR-T cell immunotherapy was performed using CAR-T cells (CD19 CAR-T cells) specific for the CD19 molecule expressed on malignant cells in B-cell leukemia and lymphoma. It has been achieved [1]-[3], and recently, using CAR-T cells (BCMA CAR-T cells) specific for B cell maturation antigen (BCMA) expressed in multiple myeloma (MM). Has been achieved [4]. Adoption of autologous or allogeneic CD19 CAR-T cells is permanent and complete in patients with acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), and MM. It induces partial response. CD19 CAR-T cells were approved by the FDA in 2017 for the treatment of relapsed / refractory ALL and NHL. Adoption of BCMA CAR-T cells induces a permanent complete and partial response in patients with MM. Numerous clinical trials using CAR-T cells targeting CD19, BCMA and other antigens are currently underway at cancer centers around the world.

Although CAR-T cell therapy has been evaluated as a remarkably powerful and highly effective new anti-cancer treatment, there are serious safety concerns. Clinical use of CAR-T cells (including CD19 CAR-T cells and BCMA CAR-T cells) reveals numerous acute and chronic, potentially life-threatening and, in some cases, lethal side effects It has so far restricted the clinical use of CAR-T cells and made it medically compatible in highly specialized cancer centers with advanced experience in bone marrow transplantation and immunotherapy. It has restricted its application. These side effects can be (but not limited to): i) strong activation and CAR- after adoption into the patient due to the presence of a large number of tumor cells expressing each target antigen. Subsequent release of cytokines from T cells (cytokine release syndrome, CRS); ii) Activation of other immune cells in the patient's body that take up tumor cell debris that accumulates as a result of tumor cell killing by CAR-T cells ( For example, macrophage activation syndrome, MAS); iii) on-target recognition and elimination of normal cells in the body of patients expressing each target antigen (eg, depletion of normal B cells by CD19 CAR-T cells); iv) Off-target recognition of normal (or malignant) cells throughout the body of a patient who does not express each target antigen of CAR; v) Due to CAR constructs, or recognition of T cells when T cells are derived from allogeneic donors. Rejection of CAR-T cells due to the immune response of the patient's immune system to the transferred CAR-T cells; vi) A motif in which CAR constructs are recognized by endogenous immune cells (eg, in the Ig-derived CAR spacer domain) Unintentional activation of CAR-T cells when harboring (Fc motif); vii) Tonic signaling and activation of CAR-T cells independent of stimulation with antigens.

A serious side effect of CAR-T cell therapy is CRS. CRS symptoms are caused by elevated levels of pro-inflammatory cytokines such as GM-CSF, IFN-γ, TNF-α, IL-2, IL-6, IL-8, IL-10 [5] and are often caused. Within hours to days of CAR-T cell transfer, it generally begins with the development of fever. CRS symptoms include tachycardia / hypotension, malaise, fatigue, myalgia, nausea, anorexia nervosa and capillary leakage, which can result in multiple organ failure [6]. The risk of developing CRS correlates with the total dose of CAR-T cells administered and the tumor load prior to CAR-T cell therapy [5], [7]. CRS is a major cause of morbidity and mortality in CAR-T cell therapy.

Currently, the ability to prevent and treat clinical CRS and to prevent or treat other side effects in the context of CAR-T cell therapy is very limited. Currently, there is no effective control of CAR-T cell function after injection into a patient. CAR-T cells are "living drugs", that is, they become part of the patient's immune system after injection into the patient, and memory CAR-T cells that grow and then shrink in the patient to prevent tumor recurrence. May last for a long time. CAR-T cell engraftment and persistence (area under the curve, AUC) has been demonstrated to correlate with therapeutic efficacy. In this regard, CAR-T cells differ from conventional drugs that are eliminated, metabolized or degraded in patients with a predictable and consistent half-life.

Currently, there are three major strategies for reducing CRS and treating or preventing the side effects of CAR-T cell therapy. 1) Tocilizumab: Interleukin-6 (IL-6) has been shown to play an essential role in CRS and therefore block IL-6R through the anti-IL-6 receptor (IL-6R) antibody tocilizumab. Has been tried in many cases and has been shown to reduce CRS in a significant proportion of patients. However, this intervention has no direct effect on CAR-T cells, but rather is a symptomatological treatment. 2) Steroids: Common attempts have been made to reduce CRS or other CAR-T cell-mediated side effects through administration of dexamethasone or prednisone. However, their ability to control CRS or other side effects is low. Since steroids are known to be immunosuppressive, their use in the context of CAR-T cell therapy raises concerns that the therapeutic effect of CAR-T cells can be negatively impacted. 3) Suicide genes and depletion markers: Some CAR-T cell products are "emergency arrest devices", ie inducible caspases that can be induced by dimerization drugs to induce apoptosis of CAR-T cells 9 It has a suicide gene such as (iCasp9). The limitation is that this strategy works well for CAR-T cells expressing high levels of this suicide gene, but with low expression [8] or antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent. It is ineffective at depletion markers such as EGFRt or CD20t, which can be induced by antibodies that induce sex cytotoxicity (CDC) and eliminate CAR-T cells. However, these antibody-dependent depletion markers can only function if the patient's immune system is unchanged, but after previous intensive chemotherapy or as part of CAR's on-target recognition. Due to the depletion of normal immune cells, this is often not the case. A major concern with suicide genes and depletion markers is that they eliminate CAR-T cells and terminate their therapeutic effect. Due to the immunogenicity of the current CAR construct, a second infusion is often not possible (because the patient develops an immune response and rejects CAR-T cells on the second infusion). Is of particular concern. As a result, there are currently no reports of cases in which iCasp9 or EGFRt was induced in the context of CAR-T cell immunotherapy.

Patients who are given CAR-T cells and are at high risk of developing CRS and / or neurotoxicity should measure serum cytokines, including but not limited to IFN-γ, IL-6, MCP1, and body temperature. It has recently been shown that it can be identified by measuring viral signs such as [2], [10]. If the function of CAR-T cells can be controlled (and intermittently rested) in such patients, it will be possible to reduce or prevent these toxicities.

Currently, for controlling the function of CAR-T cells after administration to a patient, for methods for preventing or treating CRS or other side effects while preserving the subsequent anticancer effects of CAR-T cell products. There is an unmet need.

Another challenge in CAR-T cell cancer immunotherapy is that this treatment is ineffective in a subset of patients and does not lead to the desired therapeutic response. There are several mechanisms that can lead to the ineffectiveness of CAR-T cell therapy (including but not limited to: 1) to antigens, especially in patients with high tumor burden and patients with solid tumors. CAR-T cells are exhausted by constant exposure to and consequent signal transduction from CAR. 2) CAR-T cells are exhausted after ex vivo production and subsequently do not function against engraftment, expansion, persistence, proliferation and cancer cells in the patient's body; 3) Tonics from CAR constructs CAR-T cells are exhausted due to signal transduction and cause activation-induced cell death (AICD); 4) CAR-T cells express checkpoint molecules including, but not limited to, PD-1. And the checkpoint molecule inhibits viability, proliferation and function on cancer cells. Programmed cell death protein 1 (PD-1) is expressed on the surface of T cells. PD-1 promotes apoptosis in T cells when it binds to its ligand, PD-L1, which is commonly expressed on cancer cells and in the tumor microenvironment. Blocking PD-1_PD-L1 Axis through a checkpoint blocker, an anti-PD-1 or anti-PD-L1 antibody, is a strategy for increasing the function of endogenous and CAR-modified T cells in cancer immunotherapy. It is being pursued [11].

Currently, there is an unmet need for means of improving CAR-T cell viability and function in patients who do not respond to CAR-T cell immunotherapy.

The tyrosine kinase inhibitor dasatinib (copyright Sprycel) is commonly expressed in Philadelphia chromosome-positive (Ph +) chronic myelogenous leukemia (CML) [13] and approximately 20% of cases in ALL [14]. It was developed as an inhibitor of fusion proteins. [12] Since 2010, dasatinib has been approved for first-line treatment of Ph + ALL and CML. In addition, dasatinib has been shown to block the ATP binding site of the SRC kinase Lck, which is involved in the traditional T cell signaling cascade after stimulation through the endogenous physiological T cell receptor [15] ~. [18].

J. N. Kochenderfer et al., "Lymphoma remissions caused by anti-CD19 chimeric antigen receptor T cells are associated with high serum interleukin-15 levels", J. Clin. Oncol., 2017 C. J. Turtle et al., "Immunotherapy of non-Hodgkins lymphoma with a defined ratio of CD8 + and CD4 + CD19-specific chimeric antigen receptor-modified T cells", Sci. Transl. Med., 2016 C. J. Turtle et al., "CD19 CAR-T cells of defined CD4 +: CD8 + composition in adult B cell ALL patients", J. Clin. Invest., 2016 S. A. Ali et al., "T cells expressing an anti-B-cell maturation antigen chimeric antigen receptor cause remissions of multiple myeloma.", Blood, Vol. 128, No. 13, pp. 1688-700, September 2016. M. L. Davila et al., "Efficacy and Toxicity Management of 19-28z CAR T Cell Therapy in B Cell Acute Lymphoblastic Leukemia", Sci. Transl. Med., 2014 D. W. Lee et al., "Current concepts in the diagnosis and management of cytokine release syndrome," Blood, 2014 R. J. Brentjens et al., "CD19-Targeted T Cells Rapidly Induce Molecular Remissions in Adults with Chemotherapy-Refractory Acute Lymphoblastic Leukemia", Sci. Transl. Med., P. Sc, 2013 I. Diaconu et al., "Inducible Caspase-9 Selectively Modulates the Toxicities of CD19-Specific Chimeric Antigen Receptor-Modified T Cells", Mol. Ther., Vol. 25, No. 3, pp. 580-592, March 2017. X. Wang et al., "A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells," Blood, 2011. J. Gust et al., "Endothelial Activation and Blood-Brain Barrier Disruption in Neurotoxicity after Adoptive Immunotherapy with CD19 CAR-T Cells", Cancer Discov., 2017 J. Weber, "Immune checkpoint proteins: A new therapeutic paradigm for cancerpreclinical background: CTLA-4 and PD-1 blockade", Seminars in Oncology. 2010 JS Tokarski et al., "The structure of dasatinib (BMS-354825) bound to activated ABL kinase domain elucidates its inhibitory activity against imatinib-resistant ABL mutants", Cancer Res., Vol. 66, No. 11, pp. 5790-5977, June 2006 Moon P. C. Nowell and D. A. Hungerford, "Chromosome Studies on Normal and Leukemic Human Leukocytes 1" D. Catovsky et al., "Multiparameter studies in lymphoid leukemias.", Am. J. Clin. Pathol., Vol. 72, No. 4, Suppl, pp. 736-45, October 1979. S. Blake, TP Hughes, G. Mayrhofer, and AB Lyons, "The Src / ABL kinase inhibitor dasatinib (BMS-354825) inhibits function of normal human T-lymphocytes in vitro," Clin. Immunol., Vol. 127, No. 3. , Pages 330-339, June 2008 F. Fei et al., "Dasatinib exerts an immunosuppressive effect on CD8 + T cells specific for viral and leukemia antigens", Exp. Hematol., Vol. 36, No. 10, pp. 1297-1308, October 2008 CK Fraser et al., "Dasatinib inhibits recombinant viral antigen-specific murine CD4 + and CD8 + T-cell responses and NK-cell cytolytic activity in vitro and in vivo", Exp. Hematol., Vol. 37, No. 2, pp. 256-265, 2009 February of the year R. Weichsel et al., "Profound Inhibition of Antigen-Specific T-Cell Effector Functions by Dasatinib", Clin. Cancer Res., Vol. 14, No. 8, pp. 2484-2491, March 2008 M. Kalos et al., "T Cells with Chimeric Antigen Receptors Have Potent Antitumor Effects and Can Establish Memory in Patients with Advanced Leukemia," Sci. Transl. Med., 2011 J. N. Kochenderfer et al., "Donor-derived CD19-targeted T cells cause regression of malignancy persisting after allogeneic hematopoietic stem cell transplantation," Blood, Vol. 122, No. 25, pp. 4129-4139, December 2013. K. C. Straathof et al., "An inducible caspase 9 safety switch for T-cell therapy", Blood, 2005 M. Hudecek et al., "Receptor affinity and extracellular domain modifications affect tumor recognition by ROR1-specific chimeric antigen receptor T cells", Clin. Cancer Res., 2013 F. R. Luo et al., "Dasatinib (BMS-354825) Pharmacokinetics and Pharmacodynamic Biomarkers in Animal Models Predict Optimal Clinical Exposure", Clin. Cancer Res., Vol. 12, No. 23, pp. 7180-7186, December 2006

The present invention utilizes our findings regarding the previously unknown and unexpected ability of the drug to block the function of CAR-T cells through continuous administration of the tyrosine kinase inhibitor dasatinib. Furthermore, the present invention leverages our findings regarding the previously unknown and unexpected ability of the drug to increase the function of CAR-T cells through intermittent administration of the tyrosine kinase inhibitor dasatinib.

According to the present invention, continuous administration of dasatinib provides rapid and complete blockade of CAR-T cell function. This blockade remains effective as long as CAR-T cells are continuously exposed to dasatinib at concentrations above a certain threshold. This blockade is effective in non-activated and already activated CAR-T cells. This blockade is effective in both CD8 + killer T cells as well as CD4 + helper (and regulatory) T cells. Moreover, this blockade is effective independently of the antigen specificity of CAR and of the specific design of CAR for antigen binding domains, extracellular spacer domains, and intracellular signaling and co-stimulating moieties. Moreover, this blockade is effective as long as the exposure to dasatinib is maintained, but is rapidly and completely reversible when the exposure to dasatinib is discontinued. Moreover, this blockade does not affect the viability of CAR-T cells and does not affect the ability of CAR-T cells to exert anticancer function when exposure to dasatinib is discontinued. According to the present invention, the ability of dasatinib to control the function of CAR-T cells is separate from and superior to the ability of steroids to control and inhibit the function of CAR-T cells. According to the present invention, the ability of dasatinib to block CAR-T cell function can be utilized to enhance the safety of CAR-T cell therapy, including but not limited to the prevention and treatment of CRS.

According to the present invention, intermittent administration of dasatinib can be utilized to increase the antitumor function of CAR-T cells. This increase is due to increased survival and function of CAR-T cells due to intermittent exposure to dasatinib. In addition, this increase is due to increased engraftment, proliferation and persistence of CAR-T cells due to intermittent exposure to dasatinib. Moreover, this increase is due to the excellent signaling of CAR due to intermittent exposure to dasatinib. In addition, this increase is due to decreased expression of inhibitory immune checkpoint molecules on CAR-T cells, including but not limited to PD-1, due to intermittent exposure to dasatinib. Intermittent administration includes any use of dasatinib at constant or variable length intervals where the concentration of dasatinib is not continuously higher than the concentration required to block CAR-T cell function. ..

The present invention is exemplified by the following preferred embodiments:
1. A composition for use in a method of treating cancer in a patient, wherein the composition contains a tyrosine kinase inhibitor.
In the method, the composition is administered to the patient, and the method is a method of treating cancer, including immunotherapy.
Composition.
2. The composition according to item 1 for use as specified in item 1, wherein the immunotherapy is adopted immunotherapy.
3. The composition according to item 1 or 2 for use as specified in item 1 or 2, wherein the immunotherapy is an immunotherapy using immune cells.
4. The composition according to item 3 for use as defined in item 3, wherein the immunotherapy is an immunotherapy using immune cells expressing a chimeric antigen receptor.
5. The composition of item 4 for use as defined in item 4, wherein the chimeric antigen receptor is capable of binding to the antigen.
6. The composition according to item 5 for use as specified in item 5, wherein the chimeric antigen receptor can bind to a cell surface antigen.
7. The composition according to item 5 or 6 for use as specified in item 5 or 6, wherein the antigen is a cancer antigen.
8. The antigens are CD4, CD5, CD10, CD19, CD20, CD22, CD27, CD30, CD33, CD38, CD44v6, CD52, CD64, CD70, CD72, CD123, CD135, CD138, CD220, CD269, CD319, ROR1, ROR2, SLAMF7, BCMA, αvβ3 integrin, α4β1 integrin, LILRB4, EpCAM-1, MUC-1, MUC-16, L1-CAM, c-kit, NKG2D, NKG2D ligand, PD-L1, PD-L2, Lewis-Y , CAIX, CEA, c-MET, EGFR, EGFRvIII, ErbB2, Her2, FAP, FR-a, EphA2, GD2, GD3, GPC3, IL-13Ra, Mesoterin, PSMA, PSCA, VEGFR, and FLT3. The composition according to any one of items 5 to 7 for use as specified in any one of items 5 to 7.
9. The composition of item 8 for use as defined in item 8, wherein the antigen is selected from the group consisting of CD19, CD20, CD22, CD123, SLAMF7, ROR1, BCMA, and FLT3.
10. The composition according to item 9 for use as specified in item 9, wherein the antigen is CD19.
11. The composition according to item 9 for use as specified in item 9, wherein the antigen is ROR1.
12. The composition according to item 9 for use as specified in item 9, wherein the antigen is BCMA.
13. The composition according to item 9 for use as specified in item 9, wherein the antigen is FLT3.
14. The composition according to item 9 for use as specified in item 9, wherein the antigen is CD20.
15. The composition according to item 9 for use as specified in item 9, wherein the antigen is CD22.
16. The composition according to item 9 for use as specified in item 9, wherein the antigen is CD123.
17. The composition of item 9 for use as specified in item 9, wherein the antigen is SLAMF7.
18. The composition according to any one of items 5 to 17 for use as defined in any one of items 5 to 17, wherein the cancer comprises cancer cells expressing the antigen.
19. Any one of items 4-18, wherein the chimeric antigen receptor comprises a co-stimulation domain selected from the group consisting of CD27, CD28, 4-1BB, ICOS, DAP10, NKG2D, MyD88 and OX40 co-stimulation domains. The composition according to any one of items 4-18 for use as specified in the section.
20. The composition of item 19 for use as defined in item 19, wherein the chimeric antigen receptor comprises a CD28 co-stimulating domain.
21. The composition of item 19 for use as defined in item 19, wherein the chimeric antigen receptor comprises a 4-1BB costimulatory domain.
22. The composition of item 19 for use as defined in item 19, wherein the chimeric antigen receptor comprises an OX40 co-stimulating domain.
23. The composition according to any one of items 3 to 22 for use as defined in any one of items 3 to 22, wherein the immune cell is a lymphocyte.
24. The composition according to any one of items 3 to 23 for use as defined in any one of items 3 to 23, wherein the immune cell is a B lymphocyte or a T lymphocyte.
25. The composition according to item 24 for use as defined in item 24, wherein the immune cell is a T lymphocyte.
26. The composition according to item 25 for use as specified in item 25, wherein the immune cells are CD4 + and / or CD8 + T lymphocytes.
27. The composition according to item 26 for use as specified in item 26, wherein the immune cell is a CD4 + T lymphocyte.
28. The composition according to item 26 for use as defined in item 26, wherein the immune cell is a CD8 + T lymphocyte.
29. The immune cells are CD8 + killer T cells, CD4 + helper T cells, naive T cells, memory T cells, central memory T cells, effector memory T cells, memory stem T cells, invariant T cells, NKT cells, and cytokine induction. For use as specified in any one of items 3-28, selected from the group consisting of sex killer T cells, gamma / delta T cells, natural killer cells, monospheres, macrophages, dendritic cells, and granulocytes. The composition according to any one of items 3 to 28 of.
30. The composition according to any one of items 1-29 for use as specified in any one of items 1-29, wherein the tyrosine kinase inhibitor is an Src kinase inhibitor.
31. The composition according to any one of items 1 to 30 for use as specified in any one of items 1 to 30, wherein the tyrosine kinase inhibitor is an inhibitor of kinase upstream of NFAT.
32. The composition according to any one of items 1-31 for use as defined in any one of items 1-31 wherein the tyrosine kinase inhibitor is an Lck kinase inhibitor.
33. Item 1-32 for use as defined in any one of items 1-32, wherein the tyrosine kinase inhibitor is selected from the group consisting of dasatinib, salacatinib, bosutinib, nilotinib, and PP1 inhibitors. The composition according to any one item.
34. The composition according to item 33 for use as defined in item 33, wherein the tyrosine kinase inhibitor is dasatinib.
35. The composition of item 33 for use as defined in item 33, wherein the tyrosine kinase inhibitor is bosutinib.
36. The composition of item 33 for use as specified in item 33, wherein the tyrosine kinase inhibitor is a PP1 inhibitor.
37. The composition of item 33 for use as defined in item 33, wherein the tyrosine kinase inhibitor is nilotinib.
38. The composition according to any one of items 3-37 for use as defined in any one of items 3-37, wherein the tyrosine kinase inhibitor causes inhibition of the immune cell.
39. The composition of item 38 for use as defined in item 38, wherein the inhibition is inhibition of the cell-mediated effector function of the immune cell.
40. The inhibition of the immune cells is their
I) Cell lytic activity and / or
II) Cytokine secretion and / or
III) The composition according to item 38 or 39 for use as specified in item 38 or 39, which is an inhibition of growth.
41. The composition of any one of items 38-40 for use as defined in any one of items 38-40, wherein the inhibition comprises inhibiting PD1 expression in the immune cell.
42. One or more of the inhibitions selected from the group consisting of GM-CSF, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-8, and IL-10. The composition according to any one of items 38 to 41 for use as defined in any one of items 38 to 41, comprising inhibiting the cytokine secretion of the cytokine in the immune cells.
43. In any one of items 38-42 for use as specified in any one of items 38-42, wherein the inhibition comprises inhibiting IFN-γ and / or IL-2 secretion of the immune cell. The composition described.
44. The composition of item 43 for use as defined in item 43, wherein the inhibition comprises inhibiting IFN-γ secretion of the immune cell.
45. The composition of item 43 for use as defined in item 43, wherein the inhibition comprises inhibiting IL-2 secretion of said immune cell.
46. The composition according to any one of items 38-45 for use as specified in any one of items 38-45, wherein the inhibition is a partial or complete inhibition.
47. The composition of any one of items 38-46 for use as defined in any one of items 38-46, wherein the inhibition does not reduce the viability of the immune cell.
48. The inhibition does not reduce the viability of the immune cells over a given period of time when the composition is administered to the patient, the period being 1 hour, preferably 2 hours, preferably 3 hours. 4 hours, preferably 5 hours, preferably 6 hours, preferably 8 hours, preferably 12 hours, preferably 18 hours, preferably 1 day, preferably 2 days, more preferably 3 days, even more preferably 7 Day, more preferably 2 weeks, even more preferably 3 weeks, even more preferably 4 weeks, even more preferably 2 months, even more preferably 3 months, even more preferably 6 months, item The composition according to item 47 for use as specified in 47.
49. The composition according to any one of items 38-48 for use as specified in any one of items 38-48, wherein the inhibition is reversible.
50. The composition of item 49 for use as defined in item 49, wherein the inhibition is reversed after the composition has not been administered to the patient for a given length of time.
51. The time of the given length is 3 days, preferably 2 days, more preferably 24 hours, even more preferably 18 hours, even more preferably 12 hours, even more preferably 8 hours, even more preferably. Is 6 hours, more preferably 4 hours, even more preferably 3 hours, even more preferably 2 hours, even more preferably 90 minutes, even more preferably 60 minutes, even more preferably 30 minutes, item 50. The composition according to item 50 for use as specified in.
52. The composition according to any one of items 1 to 51 for use as specified in any one of items 1 to 51, wherein the composition is administered continuously or intermittently.
53. The composition according to item 52 for use as specified in item 52, wherein the composition is administered continuously.
54. The composition of item 52 for use as specified in item 52, wherein the composition is administered intermittently.
55. The composition is administered such that after the initial administration of the composition, the serum level of the tyrosine kinase inhibitor is maintained at or above the threshold serum level during the duration of the treatment. The composition according to any one of items 1 to 54 for use as specified in any one of items 1 to 54.
56. In the method, the serum level of the tyrosine kinase inhibitor is at least once above the threshold serum level and at least once below the same threshold serum level during the duration of the treatment after the initial administration of the composition. The composition according to any one of items 1 to 55 for use as specified in any one of items 1 to 55, wherein the composition is administered so as to be maintained in.
57. The threshold serum level is in the range of 0.1 nM to 1 μM, preferably 1 nM to 500 nM, more preferably 5 nM to 100 nM, even more preferably 10 nM to 75 nM, even more preferably 25 nM to 50 nM, item 55 or The composition according to item 55 or 56 for use as specified in 56.
58. The composition of item 57 for use as specified in item 57, wherein the threshold serum level is 50 nM.
59. The threshold serum level is the minimum serum level, and at the minimum serum level, the inhibition of the immune cells is their
I) Cell lytic activity and / or
II) Cytokine secretion and / or
III) The composition according to any one of items 55-58 for use as specified in any one of items 55-58, which is a complete inhibition of growth.
60. Items 1-59 for use as defined in any one of items 1-59, wherein said treatment of cancer has improved clinical outcomes compared to said immunotherapy for said cancer alone. The composition according to any one of the above.
61. Any of items 1-60 for use as specified in any one of items 1-60, wherein the use is for reducing or preventing the toxicity associated with the immunotherapy for the cancer. The composition according to item 1.
62. For the use specified in any one of items 1-61, wherein said use is for reducing tumor burden in said patient as compared to said immunotherapy for said cancer alone. The composition according to any one of items 1 to 61.
63. In any one of items 1-62, said use in said treatment of cancer does not reduce the therapeutic effect of said immunotherapy for said cancer as compared to said immunotherapy for said cancer alone. The composition according to any one of items 1 to 62 for the prescribed use.
64. The use in said treatment of cancer is a use to increase the therapeutic effect of said immunotherapy for said cancer as compared to said immunotherapy for said cancer alone, items 1-33. The composition according to any one of items 1 to 63 for use as specified in any one.
65. The use in said treatment of cancer is a use to reduce the disease and mortality of said immunotherapy for said cancer as compared to said immunotherapy for said cancer alone, items 1-34. The composition according to any one of items 1 to 64 for use specified in any one of the following items.
66. The use in said treatment of cancer is a use to increase the antitumor efficacy of said immunotherapy for said cancer as compared to said immunotherapy for said cancer alone, items 1-. The composition according to any one of items 1 to 65 for use as specified in any one of 65.
67. The use of the immune cells in the treatment of the cancer alone as compared to the engraftment and / or persistence of the immune cells in the immunotherapy for the cancer alone in the immunotherapy for the cancer. The composition according to any one of items 3-66 for use as specified in any one of items 3-66, which is used to increase wear and / or persistence.
68. Because the use in said treatment of cancer increases the engraftment of said immune cells in said immunotherapy as compared to the engraftment of said immune cells in a method comprising said immunotherapy for said cancer alone. The composition according to any one of items 3 to 67 for use as specified in any one of items 3 to 67, which is the use of.
69. The use is to reduce the exhaustion of the immune cells in the immunotherapy for the cancer as compared to the exhaustion of the immune cells in a method comprising the immunotherapy for the cancer alone. , The composition according to any one of items 3 to 68 for use as specified in any one of items 3 to 68.
70. The composition
I) Prior to the treatment of cancer with immunotherapy and / or
II) In parallel with the aforementioned treatment of cancer with immunotherapy and / or
III) The composition according to any one of items 1 to 69 for use as specified in any one of items 1 to 69, which is administered after the treatment of cancer by immunotherapy.
71. The composition of item 70 for use as specified in item 70, wherein the composition is administered prior to said treatment of cancer by immunotherapy.
72. The composition of item 70 for use as specified in item 70, wherein the composition is administered in parallel with said treatment of cancer by immunotherapy.
73. The composition of item 70 for use as specified in item 70, wherein the composition is administered after said treatment of cancer by immunotherapy.
74. The composition of item 70 for use as defined in item 70, wherein the composition is administered prior to the treatment of cancer by immunotherapy and in parallel with said treatment of cancer by immunotherapy.
75. The composition of item 70 for use as defined in item 70, wherein the composition is administered prior to the treatment of cancer by immunotherapy and after the treatment of cancer by immunotherapy.
76. The composition of item 70 for use as defined in item 70, wherein the composition is administered in parallel with said treatment of cancer by immunotherapy and after said treatment of cancer by immunotherapy.
77. As defined in item 70, the composition is administered prior to the treatment of cancer by immunotherapy, in parallel with the treatment of cancer by immunotherapy, and after the treatment of cancer by immunotherapy. The composition according to item 70 for use in.
78. Any one of items 3 to 77 for use as defined in any one of items 3 to 77, wherein the use is a use to prevent activation of the immune cells in the immunotherapy. The composition according to.
79. The composition of item 78 for use as defined in item 78, wherein the immune cell is a dormant immune cell.
80. The composition according to any one of items 3 to 79 for use as defined in any one of items 3 to 79, wherein the immune cell is of human origin.
81. The composition of item 80 for use as defined in item 80, wherein the immune cell of human origin is a primary human cell.
82. The composition according to item 81 for use as specified in item 81, wherein the primary human cell is a primary human T lymphocyte.
83. The composition according to any one of items 80-82 for use as defined in any one of items 80-82, wherein the immune cell of human origin is an allogeneic cell with respect to the patient.
84. The composition of any one of items 80-82 for use as defined in any one of items 80-82, wherein the immune cell of human origin is a syngeneic cell with respect to the patient.
85. Any one of items 4 to 84 for use as defined in any one of items 4 to 84, wherein the immune cell is an immune cell that transiently or stably expresses the chimeric antigen receptor. The composition according to the section.
86. Any one of items 4-85 for use as specified in any one of items 4-85, wherein the chimeric antigen receptor is a first, second, or third generation chimeric antigen receptor. The composition according to paragraph 1.
87. The item for use as defined in any one of items 5-86, wherein the chimeric antigen receptor comprises a single chain variable fragment, preferably the single chain variable fragment is capable of binding to the antigen. The composition according to any one of 5 to 86.
88. Items 5-86 for use as defined in any one of items 5-86, wherein the chimeric antigen receptor comprises a ligand or fragment thereof and the ligand or fragment thereof can bind to the antigen. The composition according to any one of the above.
89. The chimeric antigen receptor consists of a group consisting of CD3 zeta, CD3 epsilon, CD3 gamma, T cell receptor alpha chain, T cell receptor beta chain, T cell receptor delta chain, and T cell receptor gamma chain. The composition according to any one of items 5 to 88 for use as specified in any one of items 5 to 88, comprising a signaling domain comprising one or more selected domains.
90. Any one of items 1-89 for use as specified in any one of items 1-89, wherein the cancer is a cancer associated with a higher risk of illness and death in the immunotherapy. The composition according to.
91. The cancer comprises cells expressing one or more checkpoint molecules, wherein the one or more checkpoint molecules are preferably A2AR, B7-H3, B7-H4, BTLA, CTLA- Items 1 to 90 for use as specified in any one of items 1 to 90, selected from the group consisting of 4, IDO, KIR, LAG3, PD-L1, PD-L2, TIM-3, and VISTA. The composition according to any one of the above.
92. The composition of item 91 for use as defined in item 91, wherein the cancer comprises cells expressing PD-L1.
93. The cancer is a cancer selected from the group consisting of carcinoma, sarcoma, myeloma, leukemia, and lymphoma, item 1-92 for use as specified in any one of items 1-92. The composition according to any one item.
94. The composition according to item 93 for use as specified in item 93, wherein the cancer is myeloma.
95. The composition according to item 93 for use as specified in item 93, wherein the cancer is leukemia.
96. The composition according to item 93 for use as specified in item 93, wherein the cancer is lymphoma.
97. For use as specified in item 93, wherein the cancer is a carcinoma, preferably a carcinoma selected from the group consisting of breast cancer, lung cancer, colorectal cancer, and pancreatic cancer. 93. The composition according to item 93.
98. The composition of item 95 for use as defined in item 95, wherein the leukemia is B-cell leukemia, T-cell leukemia, myelogenous leukemia, acute lymphoblastic leukemia, or chronic myelogenous leukemia.
99. The composition of item 96 for use as defined in item 96, wherein the lymphoma is a non-Hodgkin's lymphoma, a Hodgkin's lymphoma, or a B-cell lymphoma.
100. The cancer
I) CD19 positive and / or
II) BCMA positive and / or
III) ROR1 positive and / or
IV) FLT3 positive and / or
V) CD20 positive and / or
VI) CD22 positive and / or
VII) CD123 positive and / or
VIII) The composition according to any one of items 1-99 for use as specified in any one of items 1-99, which is a cancer characterized as SLAMF7 positive.
101. The composition according to item 100 for use as specified in item 100, wherein the cancer is CD19 positive.
102. The composition according to item 100 for use as specified in item 100, wherein the cancer is BCMA positive.
103. The composition according to item 100 for use as specified in item 100, wherein the cancer is ROR1 positive.
104. The composition according to item 100 for use as specified in item 100, wherein the cancer is FLT3 positive.
105. The composition according to item 100 for use as specified in item 100, wherein the cancer is CD20 positive.
106. The composition according to item 100 for use as specified in item 100, wherein the cancer is CD22 positive.
107. The composition according to item 100 for use as specified in item 100, wherein the cancer is CD123 positive.
108. The composition according to item 100 for use as specified in item 100, wherein the cancer is SLAMF7 positive.
109. Any one of items 1-108 for use as specified in any one of items 1-108, wherein the patient is a patient who is not eligible for the treatment of the cancer by the immunotherapy alone. The composition according to the section.
110. Item 1-109 for use as defined in any one of items 1-109, wherein the patient is not eligible for conventional adoption immunotherapy with T cells expressing the chimeric antigen receptor. The composition according to any one item.
111. The composition according to any one of items 1-110 for use as defined in any one of items 1-110, wherein the patient has an increased risk of developing cytokine release syndrome.
112. Any one of items 1-111 for use as defined in any one of items 1-111, wherein the patient has an increased risk of developing neurotoxic side effects associated with said immunotherapy. The composition according to.
113. Any of items 1-112 for use as defined in any one of items 1-112, wherein the patient has an increased risk of developing an on-target / off tumor effect associated with said immunotherapy. The composition according to paragraph 1.
114. The use specified in any one of items 1-113, wherein the patient has elevated serum levels of one or more cytokines selected from the group of IFN-γ, IL-6, and MCP1. The composition according to any one of items 1 to 113 for use.
115. For use as specified in any one of items 1-114, wherein the patient has developed an immune response to the immunotherapy and the immune response is a side effect of the immunotherapy for the cancer. The composition according to any one of items 1 to 114 of.
116. The composition according to any one of items 1-115 for use as specified in any one of items 1-115, wherein the therapeutic method is a therapeutic method in combination with allogeneic or autologous hematopoietic stem cell transplantation. Stuff.
117. The composition of any one of items 1-116 for use as defined in any one of items 1-116, wherein the composition further comprises a pharmaceutically acceptable carrier.
118. The composition according to any one of items 1 to 117 for use as specified in any one of items 1-117, wherein the composition is administered by a route other than oral administration.
119. Described in any one of items 1-118 for use prescribed in any one of items 1-118, wherein the cancer is a cancer other than chronic myelogenous leukemia and acute lymphoblastic leukemia. Composition.
120. A composition for use in a method of treating one or more side effects associated with immunotherapy in a patient, wherein the composition comprises a tyrosine kinase inhibitor.
And, in the method, the composition is administered to the patient.
Composition.
121. The composition according to item 120 for use as specified in item 120, wherein the immunotherapy is the immunotherapy specified in any one of items 2-17 and 19-29.
122. The composition according to item 120 or 121 for use as specified in item 120 or 121, wherein the cancer is a cancer as specified in any one of items 18, 90-108, and 119.
123. The composition according to item 120-122 for use as specified in items 120-122, wherein the patient is the patient specified in any one of items 109-115.
124. The composition according to items 120-123 for the use specified in items 120-123, wherein the use is the use specified in any one of items 1-119.
125. The one or more side effects associated with immunotherapy
I) Cytokine release syndrome and / or
II) Macrophage activation syndrome and / or
III) Off-target toxicity and / or
IV) On-target / off tumor recognition of normal and / or malignant cells, and / or
V) Rejection of immunotherapeutic cells and / or
VI) Unintentional activation of immunotherapeutic cells and / or
VII) Tonic signaling and activation of immunotherapeutic cells and / or
VIII) Neurotoxicity and / or
IX) The composition according to any one of items 120 to 124 for use as specified in any one of items 120 to 124, selected from the group consisting of tumor lysis syndrome.
126. The composition according to item 125 for use as defined in item 125, wherein the side effect associated with immunotherapy is cytokine release syndrome.
127. The composition according to item 125 for use as specified in item 125, wherein the side effect associated with immunotherapy is off-target toxicity.
128. The composition of item 125 for use as defined in item 125, wherein the side effect associated with immunotherapy is on-target / off tumor recognition of normal and / or malignant cells.
129. The composition of item 125 for use as defined in item 125, wherein the side effect associated with immunotherapy is rejection of immunotherapeutic cells.
130. The composition of item 125 for use as defined in item 125, wherein the side effect associated with immunotherapy is unintended activation of immunotherapeutic cells.
131. The composition of item 125 for use as defined in item 125, wherein the side effect associated with immunotherapy is tonic signaling and activation of immunotherapeutic cells.
132. The composition according to item 125 for use as specified in item 125, wherein the side effect associated with immunotherapy is neurotoxicity.
133. The composition according to item 125 for use as defined in item 125, wherein the side effect associated with immunotherapy is tumor lysis syndrome.
134. The cytokine release syndrome is one selected from the group consisting of GM-CSF, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-8, and IL-10. The composition according to item 126 for use as specified in item 126, characterized by elevated cytokine serum levels of multiple cytokines.
135. The composition of item 134 for use as specified in item 134, wherein the use is for causing a reduction in one or more of the elevated cytokine serum levels.
136. The composition according to item 134 or 135 for use as defined in item 134 or 135, wherein the cytokine release syndrome is caused by the immunotherapy.
137. A composition for use in a method of regulating cells expressing a chimeric antigen receptor in immunotherapy for treating cancer in a patient, wherein the composition comprises a tyrosine kinase inhibitor.
And, in the method, the composition is administered to the patient.
Composition.
138. The composition according to item 137 for use as specified in item 137, wherein the immunotherapy is the immunotherapy specified in any one of items 2-17, 19-29, and 125-136.
139. The composition according to item 137 or 138 for use as specified in item 137 or 138, wherein the cancer is the cancer specified in any one of items 18, 90-108, and 119.
140. The patient according to any one of items 109 to 115, according to any one of items 137 to 139 for use as specified in any one of items 137 to 139. Composition.
141. The use described in any one of items 137-140 for the use specified in any one of items 137-140, wherein the use is the use specified in any one of items 1-136. Composition.
142.I) Immune cells and
II) A composition comprising a tyrosine kinase inhibitor.
143. The composition of item 142, wherein the immune cell is the immune cell specified in any one of items 3-17, 19-29, and 79-89.
144. The composition according to item 142 or 143, wherein the tyrosine kinase inhibitor is the tyrosine kinase inhibitor specified in any one of items 30 to 59.
145. The composition according to any one of items 142 to 144, wherein the composition comprises a pharmaceutically acceptable carrier.
146. For use as specified in any one of items 1-141,
I) Immune cells and
II) Combination with tyrosine kinase inhibitor.
147. The combination according to item 144, wherein the immune cell is an immune cell as defined in any one of items 3-17, 19-29, and 79-89.
148. The combination according to item 146 or 147, wherein the tyrosine kinase inhibitor is the tyrosine kinase inhibitor specified in any one of items 30 to 59.

Figure 1: CAR construct. scFv: Single chain variable fragment (VH- (G ₄ S) ₃ linker-VL). IgG4-FC Hinge: Hinge domain of immunoglobulin G4. CD28: CD28 co-stimulation domain. 4-1BB: 4-1BB co-stimulation domain. 3 Zetas: CD3 Zeta stimulation domain. 2A: T2A ribosome skip motif. tEGFR: Cleaved epidermal growth factor receptor. (A) CAR CD19 with a 4-1BB costimulatory domain. (SEQ ID NO: 1 to SEQ ID NO: 9). The amino acid sequences (A) to (E) are represented by the one-letter notation of amino acids in the order from the N-terminal to the C-terminal. Note that the C-terminus of the amino acid sequence is represented by an asterisk. Figure 1: CAR construct. scFv: Single chain variable fragment (VH- (G ₄ S) ₃ linker-VL). IgG4-FC Hinge: Hinge domain of immunoglobulin G4. CD28: CD28 co-stimulation domain. 4-1BB: 4-1BB co-stimulation domain. 3 Zetas: CD3 Zeta stimulation domain. 2A: T2A ribosome skip motif. tEGFR: Cleaved epidermal growth factor receptor. (B) CD28 CAR CD19 with a co-stimulation domain. (SEQ ID NO: 10 to SEQ ID NO: 18). The amino acid sequences (A) to (E) are represented by the one-letter notation of amino acids in the order from the N-terminal to the C-terminal. Note that the C-terminus of the amino acid sequence is represented by an asterisk. Figure 1: CAR construct. scFv: Single chain variable fragment (VH- (G ₄ S) ₃ linker-VL). IgG4-FC Hinge: Hinge domain of immunoglobulin G4. CD28: CD28 co-stimulation domain. 4-1BB: 4-1BB co-stimulation domain. 3 Zetas: CD3 Zeta stimulation domain. 2A: T2A ribosome skip motif. tEGFR: Cleaved epidermal growth factor receptor. (C) ROR1 CAR with a 4-1BB costimulatory domain. (SEQ ID NO: 19 to SEQ ID NO: 29). The amino acid sequences (A) to (E) are represented by the one-letter notation of amino acids in the order from the N-terminal to the C-terminal. Note that the C-terminus of the amino acid sequence is represented by an asterisk. Figure 1: CAR construct. scFv: Single chain variable fragment (VH- (G ₄ S) ₃ linker-VL). IgG4-FC Hinge: Hinge domain of immunoglobulin G4. CD28: CD28 co-stimulation domain. 4-1BB: 4-1BB co-stimulation domain. 3 Zetas: CD3 Zeta stimulation domain. 2A: T2A ribosome skip motif. tEGFR: Cleaved epidermal growth factor receptor. (D) SLAMF7 CAR with a 4-1BB costimulatory domain (SEQ ID NO: 30-40). The amino acid sequences (A) to (E) are represented by the one-letter notation of amino acids in the order from the N-terminal to the C-terminal. Note that the C-terminus of the amino acid sequence is represented by an asterisk. Figure 1: CAR construct. scFv: Single chain variable fragment (VH- (G ₄ S) ₃ linker-VL). IgG4-FC Hinge: Hinge domain of immunoglobulin G4. CD28: CD28 co-stimulation domain. 4-1BB: 4-1BB co-stimulation domain. 3 Zetas: CD3 Zeta stimulation domain. 2A: T2A ribosome skip motif. tEGFR: Cleaved epidermal growth factor receptor. (E) SLAMF7 CAR with CD28 co-stimulation domain (SEQ ID NO: 41-SEQ ID NO: 51). The amino acid sequences (A) to (E) are represented by the one-letter notation of amino acids in the order from the N-terminal to the C-terminal. Note that the C-terminus of the amino acid sequence is represented by an asterisk. Figure 2: Dasatinib blocks the cytolytic activity of CD8 ⁺ CAR-T cells. The cytolytic activity of CD8 ⁺ CAR-T cells was analyzed in vitro in a bioluminescence-based cytotoxicity assay. The chart shows the cytolytic activity of CD8 ⁺ CAR-T cells in the absence of dasatinib (0 nM) and in the presence of a titrated dose of dasatinib (12.5-100 nM). Percentages of specific lysis mediated by CAR-T cells were calculated using non-CAR modified T cells as references and controls. Specific lysis was determined at 1 hour intervals up to 12 hours. The data shown are summarized data obtained in an independent experiment using CAR-T cell lines prepared from n = 3 donors. * p <0.05, ** p <0.01, *** p <0.001. A) Dasatinib blocks the cytolytic activity of CD8 ⁺ T cells expressing CD19 CAR, which has a 4-1BB costimulatory domain. Target cells in this assay: K562 / CD19. Figure 2: Dasatinib blocks the cytolytic activity of CD8 ⁺ CAR-T cells. The cytolytic activity of CD8 ⁺ CAR-T cells was analyzed in vitro in a bioluminescence-based cytotoxicity assay. The chart shows the cytolytic activity of CD8 ⁺ CAR-T cells in the absence of dasatinib (0 nM) and in the presence of a titrated dose of dasatinib (12.5-100 nM). Percentages of specific lysis mediated by CAR-T cells were calculated using non-CAR modified T cells as references and controls. Specific lysis was determined at 1 hour intervals up to 12 hours. The data shown are summarized data obtained in an independent experiment using CAR-T cell lines prepared from n = 3 donors. * p <0.05, ** p <0.01, *** p <0.001. B) Dasatinib blocks the cytolytic activity of CD8 ⁺ T cells expressing CD19 CAR, which has a CD28 co-stimulating domain. Target cells in this assay: K562 / CD19. Figure 2: Dasatinib blocks the cytolytic activity of CD8 ⁺ CAR-T cells. The cytolytic activity of CD8 ⁺ CAR-T cells was analyzed in vitro in a bioluminescence-based cytotoxicity assay. The chart shows the cytolytic activity of CD8 ⁺ CAR-T cells in the absence of dasatinib (0 nM) and in the presence of a titrated dose of dasatinib (12.5-100 nM). Percentages of specific lysis mediated by CAR-T cells were calculated using non-CAR modified T cells as references and controls. Specific lysis was determined at 1 hour intervals up to 12 hours. The data shown are summarized data obtained in an independent experiment using CAR-T cell lines prepared from n = 3 donors. * p <0.05, ** p <0.01, *** p <0.001. C) Dasatinib blocks the cytolytic activity of CD8 ⁺ T cells expressing ROR1 CAR with a 4-1BB costimulatory domain. Target cells in this assay: K562 / ROR1. Figure 3: Dasatinib blocks the production and secretion of cytokines in CD8 ⁺ CAR-T cells. CD8 ⁺ CAR-T cells were co-cultured with antigen-positive (K562 / CD19 or K562 / ROR1) target cells either in the absence of dasatinib (0 nM) or in the presence of dasatinib (6.25-100 nM). Cytokines IFN-γ and IL-2 were measured by ELISA in the supernatants obtained from these co-cultures after 20 hours of incubation. The amount of each cytokine specifically produced in response to the antigen was determined by subtracting the amount of each cytokine obtained without stimulation. The chart shows the relative amount of IFN-γ and IL-2 specifically produced in response to stimulation with antigen-positive target cells in the presence of dasatinib (the amount of cytokines released in the absence of dasatinib). On the other hand, the normalized percentage) is shown. Unless otherwise indicated, the data shown are summary data obtained in an independent experiment using CAR-T cell lines prepared from n = 3 donors. * p <0.05, ** p <0.01. A) Dasatinib blocks the production and secretion of IFN-γ (left chart) and IL-2 (right chart) in CD8 ⁺ T cells expressing CD19 CAR with a 4-1BB costimulatory domain. Figure 3: Dasatinib blocks the production and secretion of cytokines in CD8 ⁺ CAR-T cells. CD8 ⁺ CAR-T cells were co-cultured with antigen-positive (K562 / CD19 or K562 / ROR1) target cells either in the absence of dasatinib (0 nM) or in the presence of dasatinib (6.25-100 nM). Cytokines IFN-γ and IL-2 were measured by ELISA in the supernatants obtained from these co-cultures after 20 hours of incubation. The amount of each cytokine specifically produced in response to the antigen was determined by subtracting the amount of each cytokine obtained without stimulation. The chart shows the relative amount of IFN-γ and IL-2 specifically produced in response to stimulation with antigen-positive target cells in the presence of dasatinib (the amount of cytokines released in the absence of dasatinib). On the other hand, the normalized percentage) is shown. Unless otherwise indicated, the data shown are summary data obtained in an independent experiment using CAR-T cell lines prepared from n = 3 donors. * p <0.05, ** p <0.01. B) Dasatinib blocks the production and secretion of IFN-γ (left chart) and IL-2 (right chart, n = 2) in CD8 ⁺ T cells expressing CD19 CAR with the CD28 co-stimulating domain. Figure 3: Dasatinib blocks the production and secretion of cytokines in CD8 ⁺ CAR-T cells. CD8 ⁺ CAR-T cells were co-cultured with antigen-positive (K562 / CD19 or K562 / ROR1) target cells either in the absence of dasatinib (0 nM) or in the presence of dasatinib (6.25-100 nM). Cytokines IFN-γ and IL-2 were measured by ELISA in the supernatants obtained from these co-cultures after 20 hours of incubation. The amount of each cytokine specifically produced in response to the antigen was determined by subtracting the amount of each cytokine obtained without stimulation. The chart shows the relative amount of IFN-γ and IL-2 specifically produced in response to stimulation with antigen-positive target cells in the presence of dasatinib (the amount of cytokines released in the absence of dasatinib). On the other hand, the normalized percentage) is shown. Unless otherwise indicated, the data shown are summary data obtained in an independent experiment using CAR-T cell lines prepared from n = 3 donors. * p <0.05, ** p <0.01. C) Dasatinib blocks the production and secretion of IFN-γ (left chart) and IL-2 (right chart) in CD8 ⁺ T cells expressing ROR1 CAR with a 4-1BB costimulatory domain. Figure 4: Dasatinib blocks the proliferation of CD8 ⁺ CAR-T cells. CD8 ⁺ CAR-T cells labeled with CFSE and antigen-positive (K562 / CD19 or K562 / ROR1) target cells either in the absence of dasatinib (0nM) or in the presence of dasatinib (3.125-100nM) Co-cultured with. CAR-T cell proliferation was analyzed by flow cytometry after 72 hours of incubation to determine the proliferation index. The chart shows the relative proliferation in response to stimulation with antigen-positive target cells in the presence of dasatinib (percentage normalized to the proliferation index of CAR-T cells in the absence of dasatinib). The data shown are summarized data obtained in an independent experiment using CAR-T cell lines prepared from n = 3 donors. * p <0.05, ** p <0.01. A) Dasatinib blocks the proliferation of CD8 ⁺ T cells expressing CD19 CAR with a 4-1BB costimulatory domain. Figure 4: Dasatinib blocks the proliferation of CD8 ⁺ CAR-T cells. CD8 ⁺ CAR-T cells labeled with CFSE and antigen-positive (K562 / CD19 or K562 / ROR1) target cells either in the absence of dasatinib (0nM) or in the presence of dasatinib (3.125-100nM) Co-cultured with. CAR-T cell proliferation was analyzed by flow cytometry after 72 hours of incubation to determine the proliferation index. The chart shows the relative proliferation in response to stimulation with antigen-positive target cells in the presence of dasatinib (percentage normalized to the proliferation index of CAR-T cells in the absence of dasatinib). The data shown are summarized data obtained in an independent experiment using CAR-T cell lines prepared from n = 3 donors. * p <0.05, ** p <0.01. B) Dasatinib blocks the proliferation of CD8 ⁺ T cells expressing CD19 CAR, which has a CD28 co-stimulating domain. Figure 4: Dasatinib blocks the proliferation of CD8 ⁺ CAR-T cells. CD8 ⁺ CAR-T cells labeled with CFSE and antigen-positive (K562 / CD19 or K562 / ROR1) target cells either in the absence of dasatinib (0nM) or in the presence of dasatinib (3.125-100nM) Co-cultured with. CAR-T cell proliferation was analyzed by flow cytometry after 72 hours of incubation to determine the proliferation index. The chart shows the relative proliferation in response to stimulation with antigen-positive target cells in the presence of dasatinib (percentage normalized to the proliferation index of CAR-T cells in the absence of dasatinib). The data shown are summarized data obtained in an independent experiment using CAR-T cell lines prepared from n = 3 donors. * p <0.05, ** p <0.01. C) Dasatinib blocks the proliferation of CD8 ⁺ T cells expressing ROR1 CAR with a 4-1BB costimulatory domain. Figure 5: Dasatinib blocks the production and secretion of cytokines in CD4 ⁺ CAR-T cells. CD4 ⁺ CAR-T cells were co-cultured with antigen-positive (K562 / CD19) target cells either in the absence of dasatinib (0 nM) or in the presence of dasatinib (3.125-100 nM). Cytokines GM-CSF, IFN-γ, IL-2, IL-4, IL-5, IL-6 and IL-8 by multiplex cytokine assay in supernatants obtained from these co-cultures after 20 hours of incubation. Was measured. The chart shows the amount of cytokines specifically produced in response to stimulation with antigen-positive target cells. The data shown are summary data obtained in an independent experiment using CAR-T cell lines prepared from n = 2 donors. * p <0.05, ** p <0.01, *** p <0.001. A) Dasatinib blocks the production and secretion of cytokines in CD4 ⁺ T cells expressing CD19 CAR with a 4-1BB costimulatory domain. Figure 5: Dasatinib blocks the production and secretion of cytokines in CD4 ⁺ CAR-T cells. CD4 ⁺ CAR-T cells were co-cultured with antigen-positive (K562 / CD19) target cells either in the absence of dasatinib (0 nM) or in the presence of dasatinib (3.125-100 nM). Cytokines GM-CSF, IFN-γ, IL-2, IL-4, IL-5, IL-6 and IL-8 by multiplex cytokine assay in supernatants obtained from these co-cultures after 20 hours of incubation. Was measured. The chart shows the amount of cytokines specifically produced in response to stimulation with antigen-positive target cells. The data shown are summary data obtained in an independent experiment using CAR-T cell lines prepared from n = 2 donors. * p <0.05, ** p <0.01, *** p <0.001. B) Dasatinib blocks the production and secretion of cytokines in CD4 ⁺ T cells expressing CD19 CAR, which has a CD28 co-stimulating domain. Figure 6: Dasatinib blocks the function of SLAM F7 CAR-T cells. A) In vitro bioluminescence-based cytotoxicity of CD8 ⁺ SLAMF7 CAR-T cells (upper chart: having 4-1BB co-stimulating domain; lower chart: having CD28 co-stimulating domain) Analyzed in the assay. The chart shows the cytolytic activity of CD8 ⁺ CAR-T cells against K562 / SLAMF7 in the absence of dasatinib (0 nM) and in the presence of a titrated dose of dasatinib (20-100 nM). Percentages of specific lysis mediated by CAR-T cells were calculated using non-CAR modified T cells as references and controls. Specific lysis was determined at 1 hour intervals up to 14 hours. The data shown are summary data obtained in an independent experiment using CAR-T cell lines prepared from n = 2 donors. Figure 6: Dasatinib blocks the function of SLAM F7 CAR-T cells. B) CD8 ⁺ SLAMF7 CAR-T cells (having a 4-1BB co-stimulatory domain; dark gray: CD28) either in the absence of dasatinib (0 nM) or in the presence of dasatinib (20-100 nM) (Having a stimulating domain) were co-cultured with antigen-positive (K562 / SLAMF7) target cells. Cytokines IFN-γ (left chart) and IL-2 (right chart) were measured by ELISA in the supernatants obtained from these co-cultures after 20 hours of incubation. The amount of each cytokine specifically produced in response to the antigen was determined by subtracting the amount of each cytokine obtained without stimulation. The chart shows the relative amount of IFN-γ and IL-2 specifically produced in response to stimulation with antigen-positive target cells in the presence of dasatinib (the amount of cytokines released in the absence of dasatinib). On the other hand, the normalized percentage) is shown. The data shown are summary data obtained in an independent experiment using CAR-T cell lines prepared from n = 2 donors. *** p <0.001. Figure 6: Dasatinib blocks the function of SLAM F7 CAR-T cells. C) CD4 ⁺ SLAMF7 CAR-T cells (with light gray: 4-1BB co-stimulating domain; dark gray: with CD28) either in the absence of dasatinib (0 nM) or in the presence of dasatinib (20-100 nM) (Having a stimulating domain) were co-cultured with antigen-positive (K562 / SLAMF7) target cells. Cytokines IFN-γ (left chart) and IL-2 (right chart) were measured by ELISA in the supernatants obtained from these co-cultures after 20 hours of incubation. The amount of each cytokine specifically produced in response to the antigen was determined by subtracting the amount of each cytokine obtained without stimulation. The chart shows the relative amount of IFN-γ and IL-2 specifically produced in response to stimulation with antigen-positive target cells in the presence of dasatinib (the amount of cytokines released in the absence of dasatinib). On the other hand, the normalized percentage) is shown. The data shown are summary data obtained in an independent experiment using CAR-T cell lines prepared from n = 2 donors. *** p <0.001. Figure 7: Dasatinib blocks the phosphorylation of tyrosine kinases involved in CAR signaling. CD8 ⁺ T cells expressing CD19 CAR with 4-1BB co-stimulation were co-cultured with RCH-ACV target cells either in the absence of dasatinib (dasatinib-) or in the presence of dasatinib (100 nM; dasatinib +). did. Western blotting was performed to determine phosphorylation and total protein expression of Lck / Src family kinases (Y416), CAR-associated CD3 zetas (Y142), and ZAP70 (Y319). A) Phosphorylation of Src family kinases (Y416), CAR-associated CD3 zetas (Y142), ZAP70 (Y319) in dasatinib-treated vs. dasatinib-untreated T cells, and Western showing total expression of the corresponding proteins Lck, CD3 zetas and ZAP70. Blot. Beta-actin is stained as a loading control and used for normalization. Figure 7: Dasatinib blocks the phosphorylation of tyrosine kinases involved in CAR signaling. CD8 ⁺ T cells expressing CD19 CAR with 4-1BB co-stimulation were co-cultured with RCH-ACV target cells either in the absence of dasatinib (dasatinib-) or in the presence of dasatinib (100 nM; dasatinib +). did. Western blotting was performed to determine phosphorylation and total protein expression of Lck / Src family kinases (Y416), CAR-associated CD3 zetas (Y142), and ZAP70 (Y319). B) The chart shows the relative phosphorylation (percentage) in dasatinib untreated T cells (100%) versus dasatinib treated T cells. Summary data obtained by quantitative Western blot analysis in an independent experiment with n = 3. * p <0.05. Figure 8: Dasatinib blocks NFAT-mediated expression of GFP in CD8 ⁺ and CD4 ⁺ CAR-T cells. CD8 ⁺ (left panel) and CD4 ⁺ (right panel) T cells expressing CD19 CAR with 4-1BB co-stimulation were modified using an NFAT-induced GFP reporter gene. T cells were then stimulated with CD19-positive (Raji) or CD19-negative (K562) target cells for 24 hours either in the presence of dasatinib (100 nM; dasatinib +) or in the absence of dasatinib (dasatinib-). Then, the induction of the reporter gene was analyzed by flow cytometry. The chart shows the average fluorescence intensity (MFI) obtained for GFP (green fluorescent protein) in the FITC channel. The results show summary data obtained in an independent experiment with n = 3. ** p <0.01, *** p <0.001. Figure 9: Blocking with dasatinib does not reduce CAR-T cell viability. CD8 ⁺ T cells expressing CD19 CAR with 4-1BB co-stimulation in the absence of dasatinib (dasatinib-) or in the presence of dasatinib (100 nM, dasatinib +) are CD19-positive target cells (K562 / CD19). ) And co-cultured for 24 hours. In one setting, dasatinib was added to the medium 1 hour after the start of co-culture [dasatinib (+)]. At the end of co-culture, viable T cells (Annexin ^{-V - / 7-AAD -)} , apoptotic T cells (Annexin ^{-V + / 7-AAD -)} , and death T cells (Annexin -V ⁺ / 7-AAD ⁺ ) Percentage was determined by flow cytometry. The chart shows the average percentage of live, apoptotic and dead T cells obtained in an independent experiment with n = 3. * p <0.05. Figure 10: Dasatinib blocks the function of activated CD8 ⁺ CAR-T cells. A) Dasatinib blocks the cytolytic activity of activated CD8 ⁺ CAR-T cells expressing CD19 CAR with a 4-1BB costimulatory domain. As shown in Figure 2, the cytolytic activity of CD8 ⁺ CAR-T cells was analyzed in an in vitro bioluminescence-based cytotoxicity assay. Dasatinib (100 nM) was added either at the start of the cytotoxic assay (0 h) or 1 hour after the start of the cytotoxic assay (1 h). The results show summary data obtained in an independent experiment with n = 3. * p <0.05, ** p <0.01, *** p <0.001. Figure 10: Dasatinib blocks the function of activated CD8 ⁺ CAR-T cells. B) Dasatinib blocks the production and secretion of cytokines in activated CD8 ⁺ CAR-T cells. As shown in FIG. 3, cytokine production and secretion were analyzed by ELISA. Dasatinib (100 nM) was added either at the start of co-culture (0 h) or 2 hours after the start of co-culture (+ 2 h). The results show summary data obtained in an independent experiment with n = 3. * p <0.05, ** p <0.01, *** p <0.001. Figure 10: Dasatinib blocks the function of activated CD8 ⁺ CAR-T cells. C) Dasatinib blocks the proliferation of activated CD8 ⁺ CAR-T cells. As shown in FIG. 4, the growth was analyzed by diluting the CFSE dye. Dasatinib (100 nM) was added either at the start of co-culture (0 h) or 1 hour (+ 1 h), 3 hours (+ 3 h) or 48 hours (+ 48 h) after the start of co-culture. The results show summary data obtained in an independent experiment with n = 3. * p <0.05, *** p <0.001. Figure 11: Dasatinib prevents CAR-T cell activation during sequential stimulation. CD8 ⁺ (left panel) and CD4 ⁺ (right panel) T cells expressing CD19 CAR with 4-1BB co-stimulation were modified using an NFAT-induced GFP reporter gene. T cells were then stimulated every 24 hours with CD19-positive (Raji) target cells. Dasatinib was added either at the start of the assay (black circles) or 1 hour after the start of the assay (dasa + 1h, gray circles) and then added to the medium every 24 hours at the same time as new target cells. Untreated CAR T cells were included for comparison (untreated, white circles). The induction of the reporter gene was analyzed by flow cytometry. The chart shows the average fluorescence intensity (MFI) obtained for GFP (Green Fluorescent Protein) in the FITC channel. The data shown are mean + SD obtained in n = 2 (CD8 ⁺ ) and n = 3 experiments (CD4 ⁺ ) with T cells from different healthy donors. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, by two-way ANOVA. Figure 12: Blockade of CAR-T cell function is rapid and completely reversible after short-term exposure to dasatinib. Blocking of the cytolytic activity of CAR-T cells is rapid and completely reversible after a short, 2 hour exposure to dasatinib. As shown in Figure 2, the cytolytic activity of CD8 ⁺ CAR-T cells was analyzed in an in vitro bioluminescence-based cytotoxicity assay. Dasatinib (100 nM) was added at the start of the cytotoxicity assay (-2h) and then washed away (0h). CD8 ⁺ CAR-T cells not exposed to dasatinib (0 nM) served as a reference. *** p <0.001. A) Assay performed using CD8 ⁺ T cells expressing CD19 CAR with 4-1BB co-stimulation. The data shown are summary data obtained in an independent experiment with n = 3. Figure 12: Blockade of CAR-T cell function is rapid and completely reversible after short-term exposure to dasatinib. Blocking of the cytolytic activity of CAR-T cells is rapid and completely reversible after a short, 2 hour exposure to dasatinib. As shown in Figure 2, the cytolytic activity of CD8 ⁺ CAR-T cells was analyzed in an in vitro bioluminescence-based cytotoxicity assay. Dasatinib (100 nM) was added at the start of the cytotoxicity assay (-2h) and then washed away (0h). CD8 ⁺ CAR-T cells not exposed to dasatinib (0 nM) served as a reference. *** p <0.001. B) Assay performed using CD8 ⁺ T cells expressing CD19 CAR with CD28 co-stimulation. The data shown are summary data obtained in an independent experiment with n = 2. Figure 13: Long-term exposure to dasatinib does not reduce CAR-T cell viability. CD8 ⁺ T cells expressing CD19 CAR with 4-1BB co-stimulation were maintained in culture medium containing dasatinib [100 nM, (+)]. Before co-culture [d0 (-)], after two days (d2) and 8 days post (d8), viable T cells (Annexin ^{-V - / 7-AAD -)} , apoptotic T cells (Annexin -V ⁺ / 7- AAD ^-), and were determined by flow cytometry percentage of dead T cells (annexin ^{-V + / 7-AAD +)} . Untreated CD8 ⁺ CAR-T cells [(-)] were stained for comparison on the day mentioned. The chart shows the average percentage of live, apoptotic and dead T cells obtained in data from one healthy donor. Figure 14: Blocking of CAR-T cell function is rapid and completely reversible after long-term exposure to dasatinib and subsequent removal, and blocking of CAR-T cell function remains effective after long-term exposure to dasatinib. A) Blockade of CAR-T cell cytolytic activity is rapid and completely reversible after long-term exposure to dasatinib and subsequent removal, and blockade of CAR-T cell cytolytic activity remains after long-term exposure to dasatinib. It is effective. CD8 ⁺ T cells expressing CD19 CAR with 4-1BB co-stimulation were maintained in culture medium containing dasatinib (100 nM). After 1 day (left panel) and 7 days (right panel), the aliquots of CD8 ⁺ CAR-T cells were washed and their cell lysis was performed in an in vitro bioluminescence-based cytotoxic assay as shown in Figure 2. The activity was analyzed. Dasatinib was added to the co-culture to a final concentration of 100 nM at the start of the cytotoxicity assay to analyze whether blocking of cytolytic activity was still effective after long-term exposure to dasatinib. The data shown are summarized data obtained in an independent experiment using CAR-T cell lines prepared from n = 3 donors. Legendary Description: No dasa / No dasa: No exposure to dasatinib during 1 or 7 day culture, dasatinib is absent during cytotoxic assay. No dasa / dasa: No exposure to dasatinib during 1 or 7 day culture, dasatinib is present during the cytotoxic assay. dasa / no dasa: Exposed to dasatinib during 1 or 7 day culture, dasatinib is absent during cytotoxicity assay. dasa / dasa: Exposed to dasatinib during 1 or 7 day culture, dasatinib is present during the cytotoxic assay. *** p <0.001. Figure 14: Blocking of CAR-T cell function is rapid and completely reversible after long-term exposure to dasatinib and subsequent removal, and blocking of CAR-T cell function remains effective after long-term exposure to dasatinib. B) Blockade of CAR-T cell cytokine production and secretion is rapid and completely reversible after long-term exposure to dasatinib and subsequent removal, and blockade of CAR-T cell cytokine production and secretion to dasatinib. Still effective after long-term exposure. CD8 ⁺ T cells expressing CD19 CAR with 4-1BB co-stimulation were maintained in culture medium containing dasatinib (100 nM). After 1 and 7 days, the aliquots of CD8 ⁺ CAR-T cells were washed and cytokine production and secretion were analyzed as shown in FIG. Dasatinib was added to a final concentration of 100 nM at the start of co-culture to analyze whether blocking cytokine production and secretion remained effective after long-term exposure to dasatinib. The data shown are summarized data obtained in an independent experiment using CAR-T cell lines prepared from n = 3 donors. Legend Description: Before Dasa-No exposure to dasatinib during 1 or 7 day culture. Before Dasa 1: Expose to dasatinib for 1 day. Before Dasa 7: Exposure to dasatinib for 7 days. Between Dasa-: Dasatinib is not present during co-culture for cytokine assay. Between Dasa +: Dasatinib is present during co-culture for cytokine assays. *** p <0.001. Figure 14: Blocking of CAR-T cell function is rapid and completely reversible after long-term exposure to dasatinib and subsequent removal, and blocking of CAR-T cell function remains effective after long-term exposure to dasatinib. C) Blocking CAR-T cell proliferation is rapid and completely reversible after long-term exposure to dasatinib and subsequent removal, and blocking CAR-T cell proliferation remains effective after long-term exposure to dasatinib. .. CD8 ⁺ T cells expressing CD19 CAR with 4-1BB co-stimulation were maintained in culture medium containing dasatinib (100 nM). After 1 and 7 days, the aliquots of CD8 ⁺ CAR-T cells were washed and growth was analyzed as shown in FIG. Dasatinib was added to a final concentration of 100 nM at the start of co-culture to analyze whether growth blockade was still effective after long-term exposure to dasatinib. The data shown are summarized data obtained in an independent experiment using CAR-T cell lines prepared from n = 3 donors. Legend Description: Before Dasa-No exposure to dasatinib during 1 or 7 day culture. Before Dasa 1: Expose to dasatinib for 1 day. Before Dasa 7: Exposure to dasatinib for 7 days. Between Dasa-: Dasatinib is absent during co-culture for growth assay. Between Dasa +: Dasatinib is present during co-culture for growth assay. ** p <0.01, *** p <0.001. Figure 15: Dasatinib blocks cytokine secretion from CAR-T cells in vivo and prevents cytokine release syndrome. A) Experimental setting and treatment schedule: NSG mice were inoculated with firefly luciferase-transfected Raji tumor cells by iv tail intravenous injection on day 7 and dasatinib was injected by ip every 6 hours from 0 hour on day 0. It was administered until 30 hours on the first day (6 doses in total). CAR-T cells (ie, CD8 ⁺ and CD4 ⁺ CD19 CAR / 4-1BB T cells, total dose: 5 × 10e6; CD8: CD4 ratio = 1: 1) or control non-transduced T cells at day 0 3 Administered at the time. Bioluminescence imaging was performed on days 1, 1 and 3 to determine tumor loading. A cohort of mice was sacrificed at 33 hours on day 1 and on day 3 and peripheral blood (PB), bone marrow (BM) and spleen (SP) were analyzed. Figure 15: Dasatinib blocks cytokine secretion from CAR-T cells in vivo and prevents cytokine release syndrome. B) Cytokine levels in mouse serum were determined by multiplex cytokine analysis in samples obtained at 33 hours on day 1 and on day 3. The chart was treated with non-transfected control T cells and was not given dasatinib (control /-); was treated with CD19 CAR-T cells and was not given dasatinib (CAR /-); GM-CSF, IFN-γ, TNF-α, IL-2, IL-5 and IL obtained in a cohort of (CAR / +) mice treated with CD19 CAR-T cells and fed dasatinib, respectively. Indicates a concentration of -6. * p <0.05; ** p <0.01. Figure 15: Dasatinib blocks cytokine secretion from CAR-T cells in vivo and prevents cytokine release syndrome. C) Raji tumor loading was determined by bioluminescence imaging on days 1-, 1 and 3. The chart was treated with non-transduced control T cells and was not given dasatinib (control /-); was given non-transduced control T cells and dasatinib (control / +); CD19 CAR-T cells. Obtained in a cohort of mice treated with and not fed dasatinib (CAR /-); treated with CD19 CAR-T cells and fed dasatinib (CAR / +), day 1. The mean multiple changes in the bioluminescence signal between day 1 (black bar) and between day 1 and day 3 (gray bar) are shown. ** p <0.01; *** p <0.001. Figure 15: Dasatinib blocks cytokine secretion from CAR-T cells in vivo and prevents cytokine release syndrome. D) The presence of adopted CAR modified and control non-transduced T cells in peripheral blood (PB), bone marrow (BM) and spleen (Sp) was analyzed by flow cytometry on days 1 and 3. Charts, live ^- showing the frequency of (identified as human CD3 ⁺ / human CD45 ⁺⁾ CAR modifications and control non-transduced T cells as a percentage of the cells (7-AAD). Legend Description: Control / Untreated: Mice were fed non-transduced control T cells and were not fed dasatinib; Control / Treatment: Mice were fed non-transduced control T cells and were fed dasatinib CAR / untreated: mice were fed CD19 CAR-T cells and not dasatinib; CAR / treated: mice were fed CD19 CAR-T cells and were fed dasatinib. Figure 15: Dasatinib blocks cytokine secretion from CAR-T cells in vivo and prevents cytokine release syndrome. E) Adopted CD19 CAR / 4-1BB modified and non-transduced control T cells also carried an NFAT-induced GFP reporter gene. Expression of the GFP reporter gene was analyzed by flow cytometry in bone marrow (lower chart) and spleen (upper chart) in CAR-modified and control T cells. The chart shows the average fluorescence intensity (MFI) of GFP in CAR-modified and control non-transduced T cells (identified as human CD3 ⁺ / human CD45 ⁺ ). Legend Description: Control / Untreated: Mice were fed non-transduced control T cells and were not fed dasatinib; Control / Treatment: Mice were fed non-transduced control T cells and were fed dasatinib CAR / untreated: mice were fed CD19 CAR-T cells and not dasatinib; CAR / treated: mice were fed CD19 CAR-T cells and were fed dasatinib. * p <0.05; ** p <0.01; *** p <0.001. Figure 16: Dasatinib arrests activated CD19 CAR / 4-1BB-T cells in vivo in a functional off state. A) Experimental setup and treatment schedule: NSG mice were inoculated with firefly luciferase transduced Raji tumor cells by iv tail intravenous injection on day 0. Administration of CAR-T cells (ie, CD8 ⁺ and CD4 ⁺ CD19 CAR / 4-1BB T cells, total dose: 5 × 10e6; CD8: CD4 ratio = 1: 1) or control non-transduced T cells on day 7. did. Dasatinib was administered every 6 hours on days 10-12 (8 doses total) to generate functional on-off-on sequences. Bioluminescence imaging and bleeding were performed on days 7, 10, 12, 14, and 17, followed by weekly (dx) bioluminescence imaging to determine tumor loading. Figure 16: Dasatinib arrests activated CD19 CAR / 4-1BB-T cells in vivo in a functional off state. B) Progression of tumor load as measured as ventral mean luminescence over time. The upper chart shows the development of individual mice, and the lower chart shows the mean BLI of each treatment cohort. Legend Description: Controls (on / off / on): Mice were fed non-chimericated control T cells and dasatinib on days 10-12; CAR (on): Mice were fed CD19 CAR-T cells And no dasatinib was given; CAR (on / off / on): Mice were given CD19 CAR-T cells and dasatinib on days 10-12. Figure 16: Dasatinib arrests activated CD19 CAR / 4-1BB-T cells in vivo in a functional off state. C) Charts are shown with non-transduced control T cells and dasatinib (control (on / off / on)); with CD19 CAR-T cells and without dasatinib (CAR (on)); CD19 CAR -Relative changes in tumor load during the indicated days, obtained in a cohort of mice treated with T cells and dasatinib (CAR (on / off / on)). ** p <0.01; ** * p <0.001. Figure 16: Dasatinib arrests activated CD19 CAR / 4-1BB-T cells in vivo in a functional off state. D) Cytokine levels in mouse serum were determined by multiplex cytokine analysis in samples obtained on days 10, 12, 14, and 17. The chart shows the concentration of IFN-γ, and the chart on the left shows the average IFNγ and individual data points. The chart on the right shows the development of each mouse in each treatment cohort. * p <0.05; ** p <0.01. Legend Description: Controls (on / off / on): Mice were fed non-chimericated control T cells and dasatinib on days 10-12; CAR (on): Mice were fed CD19 CAR-T cells And no dasatinib was given; CAR (on / off / on): Mice were given CD19 CAR-T cells and dasatinib on days 10-12. Figure 17: Dasatinib quiesces activated CD19 CAR / CD28-T cells in vivo. A) Experimental setup and treatment schedule: NSG mice were inoculated with firefly luciferase transduced Raji tumor cells by iv tail intravenous injection on day 0. CAR-T cells (ie, CD8 ⁺ and CD4 ⁺ CD19 CAR / CD28 T cells, total dose: 5 × 10e6; CD8: CD4 ratio = 1: 1) or control non-transduced T cells were administered on day 7. Dasatinib was administered every 6 hours on days 10-12 (8 doses total) to generate functional on-off-on sequences. Bioluminescence imaging and bleeding were performed on days 7, 10, 12, 14, and 17, followed by weekly (dx) bioluminescence imaging to determine tumor loading. Figure 17: Dasatinib quiesces activated CD19 CAR / CD28-T cells in vivo. B) Progression of tumor load as measured as ventral mean luminescence over time. The chart on the left shows the median BLI of each treatment cohort, and the chart on the right shows the development of individual mice. Legend Description: CAR / Dasa: Mice were fed CD19 / CD28 CAR-T cells and dasatinib on days 10-12; CAR / DMSO: Mice were fed CD19 CAR-T cells and dasatinib Not given, but given vehicle injections on days 10-12; control / Dasa: mice were given non-chimericated control T cells and dasatinib on days 10-12; CAR /-: Mice were given CD19 CAR-T cells but not injections. Figure 17: Dasatinib quiesces activated CD19 CAR / CD28-T cells in vivo. C) The chart shows the relative changes in tumor load during the indicated days, obtained in a cohort of mice treated according to the legend. Legend Description: Control / Dasa: Mice were fed non-chimericated control T cells and dasatinib on days 10-12; CAR / Dasa: Mice were fed CD19 / CD28 CAR-T cells and days 10-12 Dasatinib was given on day; CAR / DMSO: Mice were given CD19 CAR-T cells and no dasatinib but were given vehicle injections on days 10-12; CAR /-: Mice were given CD19 CAR-T cells but not injections. ** p <0.01; *** p <0.001. Figure 18: Dasatinib exerts superior control over CAR-T cell function compared to dexamethasone. A) Dasatinib exerts superior control over cytolytic activity by CAR-T cells compared to dexamethasone. 4-1 The cytolytic activity of CD8 ⁺ T cells expressing CD19 CAR with BB co-stimulation was analyzed in an in vitro bioluminescence-based cytotoxicity assay. The chart shows the cytolytic activity of CD8 ⁺ CAR-T cells in the absence of dexamethasone (0 μM) and in the presence of a titrated dose of dexamethasone (0.1-100 μM) (see chart above). In some experiments, T cells were pretreated with indicated doses of dexamethasone for 24 hours and cytotoxicity assays were performed as described above (Chart below). The cytolytic activity of CAR-T cells in the presence of 0.1 μM dasatinib is shown for comparison with reference. Percentages of specific lysis mediated by CAR-T cells were calculated using non-specific control T cells and determined at 1 hour intervals up to 10 hours. The data shown are summarized data obtained in an independent experiment using CAR-T cell lines prepared from n = 3 donors. * p <0.05, *** p <0.001. Figure 18: Dasatinib exerts superior control over CAR-T cell function compared to dexamethasone. B) Dasatinib exerts superior control over cytokine production and secretion in CAR-T cells compared to dexamethasone. CD8 ⁺ CAR-T cells were co-cultured with antigen-positive (K562 / CD19) target cells either in the absence of dexamethasone (0 μM) or in the presence of dexamethasone (0.1-100 μM). Cytokines IFN-γ and IL-2 were measured by ELISA in the supernatants obtained from these co-cultures after 20 hours of incubation. The amount of each cytokine specifically produced in response to the antigen was determined by subtracting the amount obtained without stimulation from the amount obtained after stimulation with K562 / CD19 antigen-positive target cells. The chart shows the relative amounts (processed) of IFN-γ (upper chart, gray bar) and IL-2 (lower chart, gray bar) specifically produced in response to stimulation with antigen-positive target cells. The percentage normalized to the amount of cytokines released in the absence) is shown. In some experiments, T cells were pretreated for 24 hours with the indicated dose of dexamethasone (black bar). Cytokine production of CAR-T cells in the presence of 0.1 μM dasatinib is shown for comparison with reference. The data shown are summarized data obtained in an independent experiment using CAR-T cell lines prepared from n = 3 donors. * p <0.05, ** p <0.01. Figure 18: Dasatinib exerts superior control over CAR-T cell function compared to dexamethasone. C) Dasatinib exerts superior control over the proliferation of CAR-T cells compared to dexamethasone with respect to the proliferation of CD8 ⁺ CAR-T cells. CD8 ⁺ CAR-T cells were labeled with CFSE and co-cultured with antigen-positive (K562 / CD19) target cells either in the absence of dexamethasone (0 μM) or in the presence of dexamethasone (0.1-100 μM). .. CAR-T cell proliferation was analyzed by flow cytometry after 72 hours of incubation to determine the proliferation index. The chart shows the relative growth in response to stimulation with antigen-positive target cells (percentage normalized to the growth index of CAR-T cells in the absence of treatment) (gray bar). In some experiments, T cells were pretreated for 24 hours with the indicated dose of dexamethasone (black bar). The proliferation of CAR-T cells in the presence of 0.1 μM dasatinib is shown for comparison with reference. The data shown are summarized data obtained in an independent experiment using CAR-T cell lines prepared from n = 3 donors. *** p <0.001. Figure 19: Effect of dasatinib and other clinically approved tyrosine kinase inhibitors on CAR-T cell function. A) Cytolytic activity: The cytolytic activity of CD8 ⁺ T cells expressing ROR1 CAR with a 4-1BB co-stimulation domain was analyzed in a bioluminescence-based cytotoxicity assay. The chart shows cytolytic activity in the presence of 100 nM dasatinib, 5.3 μM imatinib, 4.2 μM lapatinib and 3.6 μM nilotinib, or untreated as a control. Percentages of specific lysis of antigen-positive target cells (RCH-ACV) mediated by CAR-T cells were calculated using non-specific control T cells as a reference and determined at 1-hour intervals up to 8 hours. The data shown are summary data obtained in an independent experiment using CAR-T cell lines prepared from n = 2 donors. Figure 19: Effect of dasatinib and other clinically approved tyrosine kinase inhibitors on CAR-T cell function. B) IFN-γ production: CD8 ⁺ CAR-T cells expressing ROR1 CAR with a 4-1BB costimulatory domain in the presence of 100 nM dasatinib, 5.3 μM imatinib, 4.2 μM lapatinib and 3.6 μM nilotinib. Alternatively, it was co-cultured with antigen-positive (RCH-ACV) target cells without treatment as a control. IFN-γ was measured by ELISA in the supernatant obtained from these co-cultures after 20 hours of incubation. The amount of IFN-γ specifically produced in response to the antigen was determined by subtracting the amount obtained without stimulation from the amount obtained using antigen-positive target cells. The data shown are summary data obtained in an independent experiment using CAR-T cell lines prepared from n = 2 donors. Figure 19: Effect of dasatinib and other clinically approved tyrosine kinase inhibitors on CAR-T cell function. C) Proliferation: CD8 ⁺ T cells expressing ROR1 CAR with a 4-1BB co-stimulation domain were labeled with CFSE and in the presence of 100 nM dasatinib, 5.3 μM imatinib, 4.2 μM lapatinib and 3.6 μM nilotinib. , Or untreated as a control, co-cultured with antigen-positive (RCH-ACV) target cells. CAR-T cell proliferation was analyzed by flow cytometry after 72 hours of incubation. The table below the histogram provides the percentage of CAR-T cells that underwent cell division, respectively ≥3 / 2/1. Figure 20: Effect of dasatinib and other Src kinase inhibitors on the cytolytic activity of CAR-T cells. The cytolytic activity of CD8 ⁺ T cells expressing CD19 CAR with a 4-1BB costimulatory domain was analyzed in a bioluminescence-based cytotoxicity assay. The chart shows the cytolytic activity of CD8 ⁺ CAR-T cells in the presence of titrated doses (1-1000 nM) of salacatinib, bosutinib, PP1 inhibitor or dasatinib. The percentage of specific lysis of antigen-positive target cells (K562 / CD19) compared to non-transduced control T cells was determined 4 hours after co-culture. Figure 21: Intermittent exposure to dasatinib increases the antitumor function of CAR-T cells in vivo. A) Experimental setup and treatment schedule: NSG mice were inoculated with firefly luciferase transduced Raji tumor cells by iv tail intravenous injection on day 0. CAR-T cells (ie, CD8 ⁺ and CD4 ⁺ T cells expressing CD19 CAR with a 4-1BB co-stimulation domain, total dose: 5 × 10e6; CD8: CD4 ratio = 1: 1) or control non-transduced T Cells were administered by iv tail intravenous injection on day 7. Dasatinib was administered by ip injection from d7 to d11 every 24 hours, and then by ip injection to d12 and d14 every 36 hours (7 doses in total). Tumor loading was determined by continuous bioluminescence imaging. On day 15, mice were sacrificed and peripheral blood (PB), bone marrow (BM) and spleen (SP) were analyzed. Figure 21: Intermittent exposure to dasatinib increases the antitumor function of CAR-T cells in vivo. B) Tumor load assessed by bioluminescence imaging. The chart shows the dorsal bioluminescent signal as mean brightness at p / s / cm ² / sr obtained from the area of interest that covers the entire dorsal body of each mouse in each treatment cohort. Each cohort consists of two animals. Legend Description: Controls /-: Mice were fed non-transduced control T cells and were not fed dasatinib; Control / +: Mice were fed non-transduced control T cells and were fed dasatinib CAR /-: Mice were fed CD19 CAR-T cells and not dasatinib; CAR / +: Mice were fed CD19 CAR-T cells and were fed dasatinib. Figure 22: Intermittent exposure to dasatinib increases CAR-T cell engraftment, proliferation and persistence in vivo. The experimental setup and treatment schedule are the same as in Figure 21A. The presence of adopted CD19 CAR modifications and control non-transduced T cells in peripheral blood (PB), bone marrow (BM) and spleen (SP) was analyzed by flow cytometry. A) Gating strategy and treatment cohort given CD19 CAR-T cells but not dasatinib (CD19 CAR, upper panel), and treatment cohort given CD19 CAR-T cells and dasatinib (lower panel) Data obtained in an exemplary mouse from. Figure 22: Intermittent exposure to dasatinib increases CAR-T cell engraftment, proliferation and persistence in vivo. The experimental setup and treatment schedule are the same as in Figure 21A. The presence of adopted CD19 CAR modifications and control non-transduced T cells in peripheral blood (PB), bone marrow (BM) and spleen (SP) was analyzed by flow cytometry. B) chart, the raw (7-AAD ^- showing the frequency of) are identified as CAR modifications and control non-transduced T cells (human CD3 ⁺ / human CD45 ⁺ as a percentage of cells). Each cohort consists of two animals. Legend Description: Controls /-: Mice were fed non-transduced control T cells and were not fed dasatinib; Control / +: Mice were fed non-transduced control T cells and were fed dasatinib CAR /-: Mice were fed CD19 CAR-T cells and not dasatinib; CAR / +: Mice were fed CD19 CAR-T cells and were fed dasatinib. Figure 23: Intermittent treatment with dasatinib reduces expression of PD1 on CAR-T cells. The experimental setup and treatment schedule are the same as in Figure 21A. The chart shows the expression of PD-1 on CD19 CAR / 4-1BB T cells as the average fluorescence intensity (MDI) obtained after staining with anti-PD1 mAb. Each cohort consists of 4 animals. ** p <0.01; *** p <0.001. Legend Description: CAR /-: Mice were fed CD19 CAR-T cells and not dasatinib (black bar); CAR / +: Mice were fed CD19 CAR-T cells and were fed dasatinib Was (gray bar). Figure 24: CAR-T cells blocked by dasatinib are susceptible to subsequent removal using the iCasp9 suicide gene. CD8 ⁺ T cells expressing CD19 CAR with 4-1BB co-stimulation were modified using the iCaspase9 suicide gene. T cells were cultured in a medium supplemented with 50 U / ml IL-2 in the absence or presence of 100 nM dasatinib and in the absence or presence of an iCaspase-inducing drug. After 24 hours, cells were labeled with anti-CD3 mAB and the presence of iCasp + T cells was analyzed by flow cytometry. The chart shows the percentage of iCasp ⁺ cells as the percentage of CAR-T cells.

Adoptive immunotherapy using genetically modified CAR-T cells is a rapidly evolving field of translational research in medicine. CD19-specific CAR-T cells have been demonstrated to induce permanent complete remission in patients with end-stage leukemia and lymphoma [2], [7], [10], [19], [20]. ]. A major concern associated with CAR-T cell therapy is the development of potentially life-threatening side effects, acute and chronic, and the infusion of these manipulated T cells into patients without control of their function and fate.

Current strategies for treating the side effects of CAR-T cell therapy are attempts to neutralize cytokines such as IL-6 associated with the clinical appearance of CRS, reducing CAR-T cell activity. The use of steroids to eliminate CAR-T cells, as well as the integration of suicide genes and depletion markers to eliminate CAR-T cells. All of these strategies have major drawbacks, and attempts to neutralize or prevent cytokine binding to receptors are symptomatic therapies that have no direct effect on the CAR-T cells themselves. , Steroids exert only incomplete regulation on CAR-T cells, cannot prevent or stop CRS and other side effects, and suicide genes and depletion markers eliminate CAR-T cells. It also terminates the antitumor effect, which is not desired by patients and physicians. None of the currently known strategies allow the patient or therapist to exert precise, on-time control over the function of CAR-T cells in the patient's body.

According to the present invention, the function of CAR-T cells is controlled in the patient's body, and the patient and the doctor exert precise and timely "remote control" on CAR-T cells after injection of CAR-T cells. Dasatinib is used to make this possible.

According to the present invention, dasatinib exerts a dose-dependent titrantable inhibitory effect on CAR signaling and subsequent CAR-T cell function. Depending on the dose of dasatinib, CAR-T cell function can be partially or completely blocked. Dasatinib-induced blockade of CAR-T cell function can be utilized to reduce or prevent toxicity in the patient's body and to control CAR-T cell function (see Example 2).

According to the present invention, functional blockade of CAR-T cells by dasatinib has a rapid and immediate onset. Blockade is complete when dasatinib is provided at concentrations above certain thresholds (ie, cytolytic activity, cytokine secretion and CAR-T cell proliferation are completely inhibited). Below this threshold, dasatinib exerts a partial blockade of CAR-T cell function. The mechanism of dasatinib-induced CAR-T cell inhibition / blocking is the interference of endogenous Src kinases such as Lck with phosphorylation and the formation and function of transcription factors such as NFAT. Blocking of transmission is included, but is not limited to (see Example 2).

According to the present invention, dasatinib can inhibit and block CAR-T cell function in both CD8 + T cells and CD4 + T cells, and is an endogenous Src kinase such as Lck, and a transcription such as NFAT. It is universally applicable to any synthetic receptor construct that uses signal transduction through factors, at least in part (see Example 2).

The present invention not only enables prevention of activation of dormant non-activated CAR-T cells, but also blocks the function of CAR-T cells that have already been activated and are exerting their effector functions (implementation). See Example 3). This is especially important given that CAR-T cells throughout the patient's body are already activated during the clinical diagnosis of clinical manifestations of CRS and side effects.

According to the present invention, the blocking effect of dasatinib on CAR-T cell function is rapid and completely reversible (see Example 5). Exposure of CAR-T cells to dasatinib does not reduce their viability and subsequently impair their ability to resume antitumor function (see Example 5). This is crucial and steroids (reducing the viability of CAR-T cells and subsequently degrading their function) (see Example 9) as well as suicide genes / depletions that terminate CAR-T cells. It is a feature that distinguishes it from markers (see Example 13). According to the present invention, dasatinib exerts complete control over all CAR-T cell functions (ie, cytolytic activity, secretion of cytokines such as IFN-γ and IL-2, proliferation), while steroids are IL. -2 Interferes only with secretion and proliferation and does not inhibit cell lysis or IFN-γ secretion (see Examples 2 and 9).

According to the present invention, the blocking effect of dasatinib on CAR-T cells is effective as long as the concentration of dasatinib is maintained above a certain threshold, and is prolonged and persisted as desired by the patient or therapist. Can be done (see Example 5).

According to the present invention, dasatinib can be used to prevent, reduce or treat side effects that occur during or after CAR-T cell therapy. In particular, dasatinib can be used to reduce, prevent and / or treat cytokine release syndrome (see Example 6).

According to the present invention, dasatinib can be administered in any manner suitable to achieve the desired concentration (eg, serum level) in the patient's body. As non-limiting examples, this includes the use of any type of pump, infusion, injection and / or oral administration.

According to the present invention, inhibition and / or blockade of CAR-T cell function may also be achieved with endogenous Src kinases such as Lck and other compounds that interfere with transcription factors such as NFAT. (See Example 10).

According to the present invention, dasatinib can also be used to increase the antitumor function of CAR-T cells. As shown in Example 11, intermittent exposure to dasatinib leads to increased survival of CAR-T cells after encounter with tumor cells. In addition, intermittent exposure of CAR-T cells to dasatinib leads to excellent survival, proliferation and persistence after adoption in vivo. In addition, intermittent exposure of CAR-T cells to dasatinib leads to superior antitumor function in vivo.

According to the invention, dasatinib can also be used to reduce the expression of checkpoint molecules on CAR-T cells, including but not limited to PD-1 (see Example 12). Therefore, the invention also includes the use of dasatinib to increase the antitumor function of CAR-T cells.

The discovery that dasatinib could interfere with CAR-T cell function and block it completely was unexpected and unpredictable. CAR is a synthetic designer receptor containing amino acid sequences and protein domains that occur in non-genetically modified human T cells. However, these amino acid sequences and domains are combined in new artificial ways, and there is currently no or very knowledge of how these domains function in CAR and generate / transmit signals. It's only limited.

The discovery that dasatinib can increase CAR-T cell function and reduce PD1 expression on CAR-T cells after intermittent exposure to dasatinib was unexpected and unpredictable. .. Rather, exposure of CAR-T cells to dasatinib would have no effect or would have been expected to exert a toxic effect.

Definitions and Embodiments Unless otherwise defined below, the terms used in the present invention will be understood in accordance with common meanings known to those of skill in the art.

The publications, patent applications, patents, and other references cited herein are incorporated in their entirety for all purposes by reference to the extent that they are consistent with the present invention. References are pointed to by reference numbers in square brackets and the corresponding reference details provided in the "References" section.

The "kinase inhibitor" referred to herein is a molecular compound that inhibits the kinase by binding to one or more kinases and exerting an inhibitory effect on the kinase. Kinase inhibitors can bind to one or more kinase species, which reduces the kinase activity of one or more kinases. Kinase inhibitors referred to herein are typically small molecules, which are small molecular weight (typically less than 1 kDa) and small size (typically smaller than 1 nm diameter) molecular. It is a compound.

In one embodiment, the kinase inhibitor is a tyrosine kinase inhibitor. In a preferred embodiment, the kinase inhibitor is a Src kinase inhibitor. In a more preferred embodiment, the kinase inhibitor is an Lck inhibitor. In a highly preferred embodiment, the kinase inhibitor is dasatinib.

The term "K _D " or "K _D value" relates to the equilibrium dissociation constant as is known in the art. In the context of the present invention, these terms may relate to the equilibrium dissociation constant of a targeting agent (eg, CAR T cell) for a particular antigen of interest (eg, CD19, ROR1, BCMA, or FLT3). The equilibrium dissociation constant is an indicator of the tendency of a complex (eg, an antigen-targeting agent complex) to reversibly dissociate into its components (eg, an antigen and a targeting agent). Methods for determining the K _D value are known in the art.

Terms such as "atyrosine kinase inhibitor" refer to the presence of a kinase inhibitor, but there are additional kinase inhibitors, such as 1, 2, 3 or more. It should be understood that it does not rule out possible possibilities. In one embodiment, according to the invention, only one kinase inhibitor is used.

In one embodiment, the chimeric antigen receptor can bind to an antigen, preferably a cancer antigen, more preferably a cancer cell surface antigen. In a preferred embodiment, the chimeric antigen receptor can bind to the extracellular domain of the cancer antigen.

In a preferred embodiment, the chimeric antigen receptor is expressed in immune cells, preferably T cells. In a preferred embodiment of the invention, the chimeric antigen receptor is expressed in T cells, and the T cells specifically bind to and grow on the cancer cells with high specificity. It makes it possible to exert an inhibitory effect, preferably a cytotoxic effect.

As used herein, "adoptive immunotherapy" refers to the transfer of immune cells into a patient for targeted treatment of cancer. The cells may originate from the patient or another individual. In adoptive immunotherapy, immune cells, preferably T cells, are typically extracted from an individual, preferably a patient, genetically modified and cultured in vitro, and administered to the patient. Adoptive immunotherapy is advantageous in that it allows for the treatment of targeted growth inhibition, preferably cytotoxicity, and less non-targeting toxicity to non-tumor cells that accompanies conventional treatment.

In a preferred embodiment according to the invention, T cells are isolated from a patient with cancer and transduced with a gene transfer vector encoding a chimeric antigen receptor capable of binding to the antigen expressed by the cancer. And is administered to the patient to treat the cancer. In a preferred embodiment, the T cells are CD8 + T cells or CD4 + T cells.

The term "intermittent administration" or "intermittent administration" in the context of a tyrosine kinase inhibitor as used herein refers to the serum level of the tyrosine kinase inhibitor in the treatment window. Refers to the use of said tyrosine kinase inhibitor in an administration regime that causes an intermittent change between the condition having and the condition in which the patient has a serum level of the tyrosine kinase inhibitor below the treatment window. The treatment window for a given tyrosine kinase inhibitor can be determined by any method known in the art. Alternatively, the term "intermittent administration" or "intermittent administration" in the context of tyrosine kinase inhibitors as used herein causes the patient to cause complete inhibition of tyrosine kinases. Intermittent changes between tyrosine kinase inhibitor serum levels and patients having tyrosine kinase inhibitor serum levels that cause partial inhibition of tyrosine kinases, or complete inhibition of tyrosine kinases by patients Intermittent changes between the condition of having serum levels of tyrosine kinase inhibitors that cause tyrosine kinase inhibitors and the condition that patients have serum levels of tyrosine kinase inhibitors that do not cause inhibition of tyrosine kinases, or the patient has partial tyrosine kinases. Tyrosine kinase inhibition in a dosing regime that causes intermittent changes between a condition with serum levels of a tyrosine kinase inhibitor that causes inhibition and a condition in which the patient has serum levels of a tyrosine kinase inhibitor that does not cause inhibition of tyrosine kinase. Refers to the use of agents. Such inhibition can be achieved by any method known in the art, eg, by measuring the activity of the tyrosine kinase itself using an appropriate enzyme assay, or by measuring cell function downstream of said kinase. Can be measured by According to the present invention, partial inhibition refers to inhibition of at least 25% up to 75% as compared to the situation in the absence of the inhibitor. As used herein, "non-inhibiting" refers to inhibition of less than 25%, preferably less than 10%, as compared to the situation in the absence of the inhibitor. According to the present invention, for T lymphocytes expressing the chimeric antigen receptor, inhibition of less than 25%, preferably less than 10%, preferably cytotoxic lysis, cytokine secretion, and proliferation of the T lymphocytes. Can be an inhibition of. According to the present invention, in the case of T lymphocytes expressing a chimeric antigen receptor, inhibition of at least 25% but 75% or less is preferably cytotoxic lysis, cytokine secretion, and proliferation of the T lymphocytes. Can be an inhibition of. According to the present invention, intermittent administration of dasatinib preferably causes an intermittent change between a state in which the serum level of dasatinib is higher than 50 nM and a state in which the serum level of dasatinib is lower than 50 nM or 50 nM. Intermittent administration preferably uses a dosing interval longer than the elimination half-life of the tyrosine kinase inhibitor, more preferably more than twice the elimination half-life of the tyrosine kinase inhibitor. Thus, even more preferably, it may be achieved by using a dosing interval that is 3 times, even more preferably 4 times, even more preferably 5 times longer than the elimination half-life of the tyrosine kinase inhibitor. For example, intermittent administration of dasatinib may be achieved preferably by using a dosing interval of at least 6 hours for dasatinib, and more preferably by using an dosing interval of at least 12 hours for dasatinib. .. It will be appreciated by those skilled in the art that for each dosing regimen, the appropriate dose of each tyrosine kinase inhibitor can be selected based on routine experiments in pharmacokinetics and pharmacodynamics.

The term "continuously administered" or "continuously administered" in the context of a tyrosine kinase inhibitor as used herein is an administration that causes complete inhibition of tyrosine kinases in a continuous manner. Refers to the use of the tyrosine kinase inhibitor in the regime. According to the present invention, complete inhibition refers to at least 75% inhibition compared to the situation in the absence of the inhibitor. Such inhibition can be achieved by any method known in the art, eg, by measuring the activity of the tyrosine kinase itself using an appropriate enzyme assay, or by measuring cell function downstream of said kinase. Can be measured by According to the present invention, in the case of T lymphocytes expressing a chimeric antigen receptor, inhibition of at least 75% can preferably be cytotoxic lysis, cytokine secretion, and inhibition of proliferation of the T lymphocytes. Alternatively, the term "continuously administered" or "continuously administered" in the context of a tyrosine kinase inhibitor as used herein is a tyrosine kinase that is continuously within the treatment window. Refers to the use of said tyrosine kinase inhibitor in a dosing regime that results in the serum level of the inhibitor. According to the present invention, continuous administration of dasatinib includes any administration in which serum levels of dasatinib are consistently maintained above 50 nM or 50 nM. In an exemplary preferred embodiment, dasatinib is administered continuously, said continuous administration comprising an oral administration of 50 mg to 200 mg every 6 to 8 hours, preferably 140 mg of dasatinib every 6 hours.

The terms "cell-mediated effector function" or "cell effector function" referred to herein describe the effect that a cell, preferably an immune cell, exerts on another cell. An exemplary "cell-mediated effector function" according to the invention is cytotoxicity in which a cell, preferably an immune cell, exerts a cytotoxic activity directed towards another cell, preferably a tumor or cancer cell. It is dissolved.

The terms "on-target / off-tumor toxicity" or "on-target / off-tumor recognition" refer to toxicity or recognition, respectively, caused by an on-target effect on non-tumor cells. Such toxicity is due to a target antigen-specific attack on a non-malignant host tissue expressing the targeted antigen, each cell of the immunotherapy, typically by the immunotherapy immune cells. It may be.

As used herein, the term "off-target toxicity" refers to a non-specific attack on a non-malignant host tissue, ie, a tissue or cell, for which immunotherapy does not express the targeted target antigen, eg, for immunotherapy. , Preferably refers to toxicity due to non-specific attack by immune cells in the immunotherapy.

As used herein, the term "macrophage activation syndrome" or "MAS" refers to excessive activation and proliferation of macrophages caused by the release of cell debris through lysis of tumor cells.

The "inhibition of cytokine secretion" referred to herein can be determined by any method known in the art. Such inhibition is preferably a reduction in cytokine serum levels, more preferably a reduction in cytokine serum levels by at least 50%.

As used herein, "cytokine release syndrome" refers to the term as it is known in the art. According to the present invention, cytokine release syndrome is the release of cytokines by immune cells, such as T lymphocytes, which can express, for example, chimeric antigen receptors in immunotherapy for cancer, and this release of cytokines in patients. Anything that causes unwanted side effects. Exemplary cytokines released by T lymphocytes that can cause the development of cytokine release syndrome in adopted immunotherapy for cancer are GM-CSF, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-8, and IL-10, preferably IFN-γ and IL2.

The terms "rejection" or "rejection of immunotherapeutic cells" are known in the art. It is preferably an immune response that occurs in a cancer patient treated with the adoptive immunotherapy for the cancer, wherein the adoptive immunotherapy is a cell surface antigen expressed in some of the cells of the cancer. Including transplantation of allogeneic or allogeneic T lymphocytes expressing a chimeric antigen receptor capable of binding to, the immune response causing depletion of said allogeneic or allogeneic T lymphocytes.

The terms "unintentional activation" or "unintentional activation of immunotherapeutic cells" as used herein are known in the art. It is preferably an adoptive immunotherapy for cancer using T lymphocytes expressing a chimeric antigen receptor capable of binding to a cell surface antigen, in which other cells, preferably immune cells, are directed to the target antigen. Independent of the specific binding of the chimeric antigen receptor, it binds to the T lymphocyte and activates the T lymphocyte in the absence of the specific antigen binding of the T lymphocyte via the chimeric antigen receptor. Refers to what causes.

The terms "tonic signaling" or "tonic signaling and activation of immunotherapeutic cells" are known in the art. It preferably refers to the activation of T lymphocytes expressing chimeric antigen receptors in adoptive immunotherapy for cancer independent of cell interactions.

As used herein, "tumor lysis syndrome" refers to the term as it is known in the art. According to the present invention, in tumor collapse syndrome, a large amount of tumor cells are lysed and lysed during immunotherapy such as adoptive immunotherapy for cancer using, for example, T lymphocytes expressing a chimeric antigen receptor. It can occur when the cell debris of tumor cells is released into the bloodstream, causing side effects associated with said immunotherapy. Release of the tumor cell debris due to cytotoxic lysis by T lymphocytes can cause, for example, renal damage.

As used herein, "neurotoxicity" refers to any process that causes a toxic effect on cells associated with the central and / or peripheral nervous system.

As used herein, "survival rate" refers to the proportion of live cells compared to dead cells. Assays for determining the proportion of living cells are known in the art. An exemplary, non-limiting method demonstrated herein includes staining with Anexin V and 7-AAD to determine the proportion of viable cells.

As used herein, the term antibody refers to any functional antibody capable of specifically binding to the antigen of interest. Although not particularly limited, the term antibody includes antibodies from birds such as chickens and any species of suitable source such as mice, goats, non-human primates and mammals such as humans. Preferably, the antibody is a humanized or human antibody. A humanized antibody is an antibody that contains a small portion of a human sequence and a non-human sequence that imparts binding specificity to the antigen of interest (eg, human FLT3). The antibody is preferably a monoclonal antibody, which can be prepared by methods well known in the art. The term antibody includes IgG-1, -2, -3, or -4, IgE, IgA, IgM, or IgD isotype antibodies. The term antibody includes monomeric antibodies (eg, IgD, IgE, IgG) or oligomeric antibodies (eg, IgA or IgM). The term antibody also includes, but is not limited to, isolated and modified antibodies such as genetically engineered antibodies such as chimeric antibodies or bispecific antibodies.

As used herein, an antibody fragment or an antibody fragment refers to a portion of an antibody that retains the ability of the antibody to specifically bind to an antigen. This ability can be determined, for example, by determining the ability of the antigen binding moiety to compete with the antibody for specific binding to the antigen by methods known in the art. Without particular limitation, the antibody fragment can be produced by any suitable method known in the art, such as recombinant DNA methods and preparation by chemical or enzymatic fragmentation of the antibody. The antibody fragment retains the ability of the Fab fragment, F (ab') fragment, F (ab') 2 fragment, single chain antibody (scFv), single domain antibody, diabody or antibody that specifically binds to the antigen. It may be any other part of the antibody.

The "antibodies" (eg, monoclonal antibodies) or "fragments thereof" described herein may be derivatized or linked to different molecules. For example, molecules that can be linked to an antibody are other proteins (eg, other antibodies), molecular labels (eg, fluorescent, luminescent, colored or radioactive molecules), drugs and / or toxic agents. The antibody or antigen binding moiety is linked either directly (eg, in the form of a fusion between two proteins) or via a linker molecule (eg, any suitable type of chemical linker known in the art). It may have been.

Terms such as "treating cancer" or "treating cancer" according to the present invention refer to therapeutic treatment. An assessment of whether a therapeutic treatment task works can be made, for example, by assessing whether the treatment inhibits cancer growth in one or more treated patients. Preferably, the inhibition is statistically significant as assessed by appropriate statistical tests known in the art. Inhibition of cancer growth can be achieved by comparing cancer growth in a group of patients treated according to the present invention to a control group of untreated patients, or by standard cancer treatment in the art and the present invention. The group of patients receiving treatment with is may be evaluated by comparing the group of patients receiving only standard cancer treatment in the art with a control group of patients. Such studies for assessing inhibition of cancer growth are designed according to approved criteria for clinical studies, eg, a double-blind, randomized study with sufficient power. The term "treating cancer" is used to mean that cancer growth is partially inhibited (ie, cancer growth in a patient is delayed compared to a patient's control group). Includes inhibition of cancer growth altogether (ie, stopping cancer growth in a patient), and inhibition of cancer growth retreating (ie, cancer shrinking). An assessment of the functioning of a therapeutic procedure can be made based on known clinical indicators of cancer progression.

Treatment of cancer according to the invention does not preclude that additional or secondary therapeutic benefits also occur in the patient. For example, an additional or secondary benefit may be enhanced engraftment of transplanted hematopoietic stem cells performed before, in parallel, or after cancer treatment.

The term "composition for use in methods of treating cancer, which is a method of treating cancer, including immunotherapy" can relate to situations in which the composition has a direct effect on cancer. Alternatively, it can relate to situations in which the composition has an indirect effect on cancer, for example by enhancing immunotherapy. For example, a composition comprising a tyrosine kinase inhibitor such as dasatinib may enhance adoptive immunotherapy, eg, adoptive immunotherapy with CAR T cells.

The treatment of cancer according to the present invention can be first-line therapy, second-line therapy, third-line therapy, or force-line therapy. Treatment can also be after forceline therapy. The meaning of these terms is known in the art and follows the terminology commonly used by the National Cancer Institute.

As used herein, the term "capable of binding" refers to the ability to form a complex with a molecule to which it is bound (eg, CD19, FLT3, BCMA, or ROR1). Bonds typically occur non-covalently by intermolecular forces such as ionic bonds, hydrogen bonds and van der Waals forces, and are typically reversible. Various methods and assays for determining binding capacity are known in the art. Bond is usually a bond with high affinity, affinity as measured by K _D values is preferably less than 1 [mu] M, more preferably less than 100 nM, more preferably less than 10 nM, even more preferably less than 1 nM, It is even more preferably less than 100 pM, even more preferably less than 10 pM, and even more preferably less than 1 pM.

As used herein, each occurrence of terms such as "comprising" or "comprises" optionally consists of "consisting of" or "consists". It may be replaced with of).

Pharmaceutically acceptable carriers, such as any suitable diluent, can be used herein as is known in the art. As used herein, the term "pharmaceutically acceptable" is approved by a federal or state regulatory body for use in mammals, and more particularly in humans, or in the United States Pharmacopeia. Means listed in the United States, European Pharmacopoeia or other generally recognized Pharmacopoeia. Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffers, and combinations thereof. It will be appreciated that the formulation is properly adapted to suit the method of administration.

The compositions and formulations according to the invention are prepared according to known standards for the preparation of pharmaceutical compositions and formulations. For example, compositions and formulations are prepared in a manner that can be adequately stored and administered, for example by using pharmaceutically acceptable ingredients such as carriers, excipients or stabilizers. Such pharmaceutically acceptable ingredients are not toxic in the amounts used when administering the pharmaceutical composition or formulation to the patient. The pharmaceutically acceptable component added to the pharmaceutical composition or formulation is the chemistry of the tyrosine kinase inhibitor present in the composition or formulation (whether the targeting agent is, for example, an antibody or fragment thereof. , Or the cell expressing the chimeric antigen receptor), may depend on the particular intended use and route of administration of the pharmaceutical composition.

In a preferred embodiment according to the invention, the composition or formulation is suitable for administration to humans, preferably the formulation is sterile and / or non-pyrogenic.

The "combination" of immune cells and tyrosine kinase inhibitors for use according to the invention is not limited to a particular mode of administration. Immune cells and tyrosine kinase inhibitors can be administered, for example, separately but simultaneously or simultaneously in one composition, or they can be administered separately at different time points.

A preferred embodiment is the use of an Src kinase inhibitor in combination with said adoptive immunotherapy to treat, reduce or prevent side effects associated with adoptive immunotherapy. A more preferred embodiment is an Src kinase inhibitor combined with said adoptive immunotherapy for cancer to treat, reduce or prevent side effects associated with adoptive immunotherapy for cancer, preferably dasatinib, salacatinib, bostinib, nirotinib, Alternatively, an immune cell, preferably a T lymphocyte, that uses a PP1 inhibitor and the adoptive immunotherapy for cancer expresses a chimeric antigen receptor that recognizes an antigen expressed by some of the cancer cells. Use, including transplantation of. An even more preferred embodiment is the use of dasatinib to treat, reduce or prevent side effects associated with adoptive immunotherapy for cancer using T lymphocytes genetically modified to express a chimeric antigen receptor. , A use in which a chimeric antigen receptor can bind to a cell surface antigen expressed in some of the cancer cells.

A preferred embodiment is the use of an Src kinase inhibitor, preferably dasatinib, salacatinib, bostinib, nilotinib, or PP1 inhibitor, most preferably dasatinib, for treating, alleviating or preventing side effects associated with adoptive immunotherapy for cancer. The immunotherapy comprises transplantation of T lymphocytes genetically modified to express a chimeric antigen receptor capable of binding to a cell surface antigen expressed in some of the cancer cells. , Use. In this embodiment, the chimeric antigen receptor expressed in the transplanted T lymphocytes binds to the cell surface antigen of the cancer cell, which causes cytotoxic lysis of the cancer cell and the adoptive immunity. Side effects associated with therapy are primarily or partially caused by the release of cell debris from the cancer cells during cytotoxic lysis mediated by the T lymphocytes expressing the chimeric antigen receptor. In a more preferred embodiment, the side effects associated with the adoptive immunotherapy caused by the release of cell debris can be classified as tumor lysis syndrome or macrophage activation syndrome.

A preferred embodiment is the use of an Src kinase inhibitor, preferably dasatinib, salacatinib, bostinib, nilotinib, or PP1 inhibitor, most preferably dasatinib, for treating, alleviating or preventing side effects associated with adoptive immunotherapy for cancer. The immunotherapy comprises transplantation of T lymphocytes genetically modified to express a chimeric antigen receptor capable of binding to a cell surface antigen expressed in some of the cancer cells. , Use. In this embodiment, the chimeric antigen receptor expressed in the transplanted T lymphocytes binds to the cell surface antigen of the cancer cells, which causes cytotoxic lysis of the cancer cells and activation of the T lymphocytes. The side effects associated with the adoptive immunotherapy are mainly or partially cytokines by the T lymphocytes expressing the chimeric antigen receptor upon binding of the chimeric antigen receptor to the cell surface antigen. Caused by the release of, preferably the cell surface antigen is on the surface of cancer cells. In a more preferred embodiment, the side effects associated with the immunotherapy caused by the release of cytokines by the T lymphocytes expressing the chimeric antigen receptor can be classified as cytokine release syndrome.

In a preferred embodiment, the use of said Src kinase inhibitor, preferably dasatinib, salacatinib, bosutinib, nilotinib, or PP1 inhibitor, most preferably dasatinib, to prevent side effects associated with adoptive immunotherapy for cancer is adopted. Includes administration of the Src kinase inhibitor prior to immunotherapy. In another preferred embodiment, the Src kinase inhibitor, preferably dasatinib, salacatinib, bosutinib, nilotinib, or PP1 inhibitor, most preferably dasatinib, for treating or alleviating the side effects associated with adoptive immunotherapy for cancer. Use comprises administration of the Src kinase inhibitor after adoptive immunotherapy for cancer, preferably when symptoms of side effects associated with said adoptive immunotherapy for cancer occur. Symptoms of side effects associated with adoptive immunotherapy for cancer may include elevated serum levels of IFN-γ, IL-6, or MCP1 and / or elevated body temperature.

In a preferred embodiment, the side effects associated with adoptive immunotherapy for cancer are predominantly or partially GM-CSF, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL- 8 or due to elevated serum levels of IL-10, preferably due to elevated serum levels of IFN-γ and IL-2. In a preferred embodiment, the method of treating cancer is adoption with allogeneic or syngeneic T lymphocytes expressing a chimeric antigen receptor capable of binding to a cell surface antigen expressed by some of the cells of the cancer. Including immunotherapy. In this embodiment, when the T lymphocyte binds to the cell surface antigen, the cytokines GM-CSF, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-8, or IL -10, preferably release IFN-γ and IL-2, causing an increase in their serum levels. A preferred embodiment is an Src kinase inhibitor for reducing the release of the cytokine by inhibiting the T lymphocytes, causing a reduction in symptoms associated with said elevation of serum levels of the cytokine, preferably dasatinib, salachanib. The use of bosutinib, nilotinib, or PP1 inhibitor, most preferably dasatinib.

In another preferred embodiment, the side effects associated with adoptive immunotherapy for cancer are primarily or partially due to on-target / off tumor recognition. In a preferred embodiment, the method of treating cancer is adoption with allogeneic or syngeneic T lymphocytes expressing a chimeric antigen receptor capable of binding to a cell surface antigen expressed by some of the cells of the cancer. Including immunotherapy. In this embodiment, the T lymphocytes bind to the cell surface antigen expressed in some of the non-tumor non-malignant cells, causing unwanted cytotoxic lysis of the non-tumor non-malignant cells. A preferred embodiment is an Src kinase inhibitor for reducing on-target / off-tumor recognition by inhibiting the cytolytic activity of said T lymphocytes, causing a reduction in symptoms associated with said on-target / off tumor recognition, preferably. Is the use of dasatinib, salacatinib, bosutinib, nilotinib, or PP1 inhibitor, most preferably dasatinib. An exemplary embodiment is the use of dasatinib in a method of treating CD19-positive cancer using T lymphocytes expressing a chimeric antigen receptor capable of binding to CD19, wherein the T lymphocytes produce CD19. It is a use that binds to the expressed non-tumor cells and leads to cytotoxic lysis of the non-tumor cells, causing unwanted on-target / off-tumor side effects in the patient.

A preferred embodiment is an Src kinase inhibitor in a method of treating cancer by adoptive immunotherapy using the T lymphocytes for inhibiting the cell-mediated effector function of T lymphocytes expressing the chimeric antigen receptor, preferably. Is the use of dasatinib, salacatinib, bostinib, nilotinib, or PP1 inhibitors, most preferably dasatinib. In a preferred embodiment, the Src kinase inhibitor causes cytokine secretion, cytotoxic lysis, or decreased proliferation of the T lymphocytes. In a preferred embodiment, the T lymphocytes GM-CSF, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-8, or IL-10, preferably IFN-γ and The cytokine secretion of IL-2 is at least 10%, 20%, 30%, 40% after administration of the Src kinase inhibitor as compared to the secretion of the cytokine in the absence of the Src kinase inhibitor. Or 50% reduction. In a preferred embodiment, the cytokine secretion is reduced by at least 50%.

In a preferred embodiment, Src in a method of treating the cancer by adoptive immunotherapy using T lymphocytes expressing a chimeric antigen receptor capable of binding to a cell surface antigen expressed in some of the cells of the cancer. The use of kinase inhibitors does not significantly reduce the viability of the T lymphocytes. In a preferred embodiment, the viability of T lymphocytes expressing the chimeric antigen receptor is at least 50%, 60%, 70%, 80%, or 90% after administration of the Src kinase inhibitor. In a preferred embodiment, the viability of T lymphocytes expressing the chimeric antigen receptor is at least 80% after administration of the Src kinase inhibitor.

In a preferred embodiment, Src in a method of treating the cancer by adoptive immunotherapy using T lymphocytes expressing a chimeric antigen receptor capable of binding to a cell surface antigen expressed in some of the cells of the cancer. The use of kinase inhibitors inhibits the proliferation of the T lymphocytes. In a preferred embodiment, proliferation of T lymphocytes expressing the chimeric antigen receptor is compared to proliferation of said T lymphocytes in the absence of the Src kinase inhibitor after administration of the Src kinase inhibitor. Reduced by at least 10%, 20%, 30%, 40% 50%, 60%, 70%, 80%, or 90%. In a preferred embodiment, the proliferation of T lymphocytes expressing the chimeric antigen receptor is reduced by at least 50% after administration of the Src kinase inhibitor.

In a preferred embodiment, Src in a method of treating the cancer by adoptive immunotherapy using T lymphocytes expressing a chimeric antigen receptor capable of binding to a cell surface antigen expressed in some of the cells of the cancer. The use of kinase inhibitors inhibits the ability of the T lymphocytes for cytotoxic lysis of target cells expressing the cell surface antigen. In a preferred embodiment, cytotoxic lysis of T lymphocytes expressing the chimeric antigen receptor is administered by the Src kinase inhibitor as compared to proliferation of the T lymphocytes in the absence of the Src kinase inhibitor. After being reduced by at least 10%, 20%, 30%, 40% 50%, 60%, 70%, 80%, or 90%. In a preferred embodiment, cytotoxic lysis of T lymphocytes expressing the chimeric antigen receptor is reduced by at least 90% after administration of the Src kinase inhibitor.

In a preferred embodiment, Src in a method of treating cancer by adoptive immunotherapy using T lymphocytes expressing a chimeric antigen receptor capable of binding to a cell surface antigen expressed in some of the cells of the cancer. The use of kinase inhibitors inhibits the ability of said T lymphocytes for the expression of PD1. In a preferred embodiment, the expression of PD1 in the T lymphocytes is statistically significantly reduced as compared to the expression of PD1 in the T lymphocytes in the absence of the Src kinase inhibitor. In a preferred embodiment, the expression of PD1 in said T lymphocytes is reduced by at least 5%, 10%, 15%, 20%, or more. In a preferred embodiment, the expression of PD1 in the T lymphocytes is reduced by at least 10%.

A preferred embodiment is the use of an Src kinase inhibitor in combination with an adoptive immunotherapy to improve or reinforce adoptive immunotherapy, wherein the Src kinase inhibitor is administered intermittently. A more preferred embodiment is an Src kinase inhibitor in combination with said adoptive immunotherapy for cancer to improve the anti-cancer effect of adoptive immunotherapy against cancer, preferably dasatinib, salacatinib, bostinib, nirotinib, or PP1 inhibition. In the use of a drug, the adopted immunotherapy for cancer involves transplantation of immune cells, preferably T lymphocytes, that express a chimeric antigen receptor that recognizes an antigen expressed by some of the cancer cells. It contains and is used, wherein the Src kinase inhibitor is administered intermittently. An even more preferred embodiment is the use of dasatinib to improve the anticancer effect of adoptive immunotherapy on cancer using T lymphocytes genetically modified to express the chimeric antigen receptor, the chimeric antigen. It is a use in which the receptor can bind to a cell surface antigen expressed in some of the cells of the cancer and dasatinib is administered intermittently. In this embodiment, dasatinib is administered intermittently, whereby the T lymphocytes are partially inhibited. The partial inhibition may be inhibition of the cell-mediated effector function of the T lymphocytes, the inhibition being at least 25% to maximum of the cell-mediated effector function of one or more of the T lymphocytes. Inhibition up to 75%. In a preferred embodiment, dasatinib is administered intermittently so that the serum level of dasatinib does not continuously rise above 50 nM or 50 nM. In another preferred embodiment, dasatinib is administered intermittently so that serum levels of dasatinib do not continuously rise above 10 nM or 10 nM. In an exemplary embodiment, dasatinib is administered intermittently, the intermittent administration comprising oral administration of 50-200 mg, preferably 100 mg of dasatinib per day.

In a preferred embodiment, the tyrosine kinase inhibitor is an Src kinase inhibitor. In a more preferred embodiment, the tyrosine kinase inhibitor is dasatinib, salacatinib, bosutinib, nilotinib, or PP1 inhibitor. In a more preferred embodiment, the inhibitor is bosutinib. In a more preferred embodiment, the inhibitor is salacatinib. In a more preferred embodiment, the inhibitor is nilotinib. In a more preferred embodiment, the inhibitor is a PP1 inhibitor. In a more preferred embodiment, the inhibitor is dasatinib.

In a preferred embodiment, the method of treating cancer is adoption with allogeneic or syngeneic T lymphocytes expressing a chimeric antigen receptor capable of binding to a cell surface antigen expressed in some of the cells of the cancer. Including immunotherapy. In a more preferred embodiment, the chimeric antigen receptors are CD4, CD5, CD10, CD19, CD20, CD22, CD27, CD30, CD33, CD38, CD44v6, CD52, CD64, CD70, CD72, CD123, CD135, CD138, CD220, CD269, CD319, ROR1, ROR2, SLAMF7, BCMA, αvβ3 integrin, α4β1 integrin, LILRB4, EpCAM-1, MUC-1, MUC-16, L1-CAM, c-kit, NKG2D, NKG2D ligand, PD-L1, PD -L2, Lewis-Y, CAIX, CEA, c-MET, EGFR, EGFRvIII, ErbB2, Her2, FAP, FR-a, EphA2, GD2, GD3, GPC3, IL-13Ra, Mesoterin, PSMA, PSCA, VEGFR, or Can bind to FLT3. In a more preferred embodiment, the chimeric antigen receptor can bind to CD19, BCMA, ROR1, FLT3, CD20, CD22, CD123, or SLAMF7.

In a preferred embodiment, the chimeric antigen receptor comprises a CD27, CD28, 4-1BB, ICOS, DAP10, NKG2D, MyD88 or OX40 co-stimulating domain. In a more preferred embodiment, the chimeric antigen receptor comprises a CD28, 4-1BB, or OX40 co-stimulating domain.

In a preferred embodiment, the chimeric antigen receptor is a CD3 zeta, CD3 epsilon, CD3 gamma, T cell receptor alpha chain, T cell receptor beta chain, T cell receptor delta chain, and T cell receptor gamma chain signaling. Includes domain.

The present invention will be illustrated by the following non-limiting examples.

(Example 1)
Materials and Methods Human Subjects Blood samples were obtained from healthy donors who provided written informed consent to participate in a study protocol approved by the Study Review Board of the University of Würzburg [Universitatsklinikum Wurzburg, Germany (UKW)]. Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation in Ficoll-Hypaque (Sigma, St. Louis, MO).

Cell line
293T, K562, Raji and RCH-ACV cell lines were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). K562-ROR1 was generated by transduction of the wrench virus of the full-length human ROR1 gene. K562-CD19 was generated by transduction of the wrench virus of the full-length human CD19 gene. Flow cytometry (GFP) in mice, organisms by transfecting K562, Raji and RCH-ACV cell lines with a lentiviral vector encoding a firefly luciferase (ffluc) -enhanced green fluorescent protein (GFP) transduction gene. It enabled detection by luminescence-based cytotoxicity assay (ffLuc) and bioluminescence imaging (ffLuc). Each cell line was cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 100 U / ml penicillin / streptomycin.

Immunophenotyping Analysis PBMC and T using one or more of the following conjugated mAbs: CD3, CD4, CD8, CD45RA, CD45RO, CD62L, PD-1 and matched isotype controls (BD Biosciences, San Jose, CA): The cell line was stained. In-house biotinylated (EZ-Link ™ Sulfo-NHS-SS-Biotin, Thermo Fisher Scientific, IL; manufacturer's instructions) anti-EGFR antibody (ImClone Systems Inc.) and streptavidin-PE (BD) CAR transfection (ie, EGFRt ⁺ ) T cells were detected by staining with Biosciences). Staining with 7-AAD (BD Biosciences) was performed to distinguish between live and dead cells as directed by the manufacturer. Flow analysis was performed on FACS Canto and the data was analyzed using FlowJo software (Treestar, Ashland, OR).

Vector construction
Construction of an epHIV7 lentiviral vector containing ROR1 or CD19-specific CAR with a 4-1BB or CD28 co-stimulating domain has been described. See reference [5], which is incorporated herein by reference in its entirety for all purposes. Schematic designs of CAR structures are provided in Figures 1A-C. All vectors contained the truncated epidermal growth factor receptor (EGFRt) encoded in the transgene cassette downstream of CAR. See reference [9], which is incorporated herein by reference in its entirety for all purposes. CAR and EGFRt transgenes were isolated by the T2A ribosome skip element.

We abruptly leave residues 422-461 of wild-type green fluorescent protein (NFAT-induced GFPwt) or mouse ornithine decarboxylase with an in vivo half-life of approximately 4 hours under the control of an NFAT-responsive element. A reporter gene vector was developed from an epHIV7 lentiviral vector containing a GFP variant (NFAT-induced GFPd4) destabilized by a mutant version.

We have constructed an inducible suicide switch containing the iCasp9 suicide gene as described in [21].

Wrench virus preparation
The supernatant of the lentivirus encoding CAR / EGFRt, ffluc / GFP and NFATindGFP was used with the Calphos transfection reagent (Clontech, Mountain View, CA) for each lentiviral vector plasmid and packaging vector pCHGP-2, It was produced in 293T cells co-transfected with pCMV-Rev2 and pCMV-G. The medium was changed 16 hours after transfection and lentivirus was recovered 72 hours later. Ultracentrifugation was performed at 4 ° C. at 24,900 rpm for 2 hours to recover the virus particles. An increased amount of virus was transduced into Jurkat cells for titration of lentivirus, and 3 days after transduction, the cells were analyzed for protein surface expression using flow cytometry.

Preparation of CAR-T cells
CAR-T cells were generated as described in [5] and [22]. Briefly, CD8 ⁺ central memory and CD4 ⁺ bulk T cells were purified from healthy donor PBMCs using negative isolation with immunomagnetic beads (Miltenyi Biotec, Bergisch-Gladbach, Germany) and bead producers. The cells were activated using anti-CD3 / CD28 beads (Life Technologies) according to the instructions in the above, and transduced using the supernatant of lentivirus at a multiplicity of infection (MOI) of 5. In some experiments, combined transduction into T cells was performed using supernatants of lentivirus encoding CAR / EGFRt and NFA TindGFP. Lentivirus transduction was performed 1 day after bead stimulation by spinocuration. T cells were propagated and maintained in RPMI-1640 containing 10% human serum, GlutaminMAX (Life Technologies), 100 U / mL penicillin-streptomycin and 50 U / mL IL-2. Trypan blue staining was performed to quantify viable T cells. After bead removal on day 6 and expansion on days 10-14, T cells were concentrated for EGFRt and rapidly expanded and proliferated protocol (ROR1 CAR-T cells and corresponding non-transduced control T cells) or irradiated CD19 ^+. Further expansion was performed using any of the antigen-specific expansions using feeder cells (CD19 CAR-T cells and corresponding non-transduced control T cells).

Analysis of CAR-T cell function Cytotoxicity: Stable transduction of ffluc_GFP into target cells with effector T cells at a 5: 1 effector-to-target (E: T) ratio at 1 × 10 ⁴ cells / well 3 Incubated in ream wells. A D-luciferin substrate (Biosynth, Staad, Switzerland) was added to the co-culture to a final concentration of 0.15 mg / ml to reduce the luminescence signal in wells containing target cells and T cells with a luminometer (Tecan, Mannedorf). , Switzerland). Specific dissolutions were calculated using standard formulas.

Cytokine secretion: 5 × 10 ⁴ T cells plated with target cells (K562 / ROR1, K562 / CD19, RCH-ACV) in a 4: 1 E: T ratio into 2 or 3 wells and IFN -γ and IL-2 were measured by ELISA, or cytokine panels were measured by multiplex cytokine immunoassay (Luminex) in supernatants removed after 20 h incubation. Specific cytokine production was calculated by subtracting the amount of cytokines released by unstimulated CAR-T cells from the amount of cytokines released after antigen-specific stimulation. Residual cytokine secretion at% as shown in the chart is normalized to CAR-T cells (100%) in the absence of dasatinib treatment.

Proliferation: T cells labeled with 0.2 μM carboxyfluorescein succinimidyl ester (CFSE, Invitrogen), washed and irradiated (80 Gy) stimulated cells at an E: T ratio of 4: 1 (K562 / ROR1, K562 / ROR1, Plated into 2 or 3 wells with K562 / CD19 or RCH-ACV). No exogenous cytokines were added to the culture medium. After 72 hours of incubation, cells were labeled with anti-CD3 mAb and analyzed by flow cytometry to assess T cell division. The proliferation index was calculated using FlowJo Software (FlowJO, LLC, Ashland, Oregon, USA) for "residual proliferation", ie, for CAR-T cell proliferation (100%) in the absence of dasatinib. Used to determine what was normalized.

Western blot analysis After expansion, T cells were washed and cultured for 2 days in the absence of exogenous IL-2. Proteins were isolated after 30 minutes of stimulation of T cells with RCH-ACV (4: 1 E: T ratio). According to the manufacturer's instructions, the following antibodies: Anti-pSrc fam Y416 (cell signaling # 2101S), Anti-Lck (cell signaling # 2752S), Anti-pCD247 Y142 (CD3 Zeta, BD # 558402), Anti-CD247 (Sigma Life) Western blots were performed under reducing conditions using science # HPA008750), anti-pZap70 Y319 (cell signaling # 2717S) and anti-Zap70 (...). Staining for β-actin was used as a loading control for normalization. Western blots were developed using a ChemiDoc MP imaging system (Biorad, Munich, Germany) and quantitative analysis of Western blots was performed using Image Lab Software (Biorad, Munich, Germany).

NFAT Reporter Assay
T cells expressing (co) the NFAT-induced GFPwt reporter gene were co-cultured at a 5: 1 E: T ratio with (80 Gy) Raji or K562 tumor cells or in the presence of 10 U IL-2 without target cells. It was cultured. T cells and target cells were co-cultured in the absence of dasatinib or in the presence of 100 nM dasatinib. After 24-hour co-culture, cells were labeled with anti-CD3 mAb and analyzed by flow cytometry to evaluate GFP expression in T cells.

Apoptosis assay
CD8 ⁺ CD19 CAR-T cells were cultured alone in the presence of 50 U of IL-2 or with irradiated (80 Gy) K562 / CD19 tumor cells at a 4: 1 E: T ratio. Dasatinib was added to a final concentration of 100 nM either at the start of the assay or 2 hours after the start of the assay. For 24-hour co-culture, label the co-culture with anti-CD8 mAb, 7AAD and Anexin V (BD Biosciences, Heidelberg, Germany) according to the manufacturer's instructions and analyze by flow cytometry for apoptosis and death. The amount of T cells was evaluated.

Removal of ICasp + T cells
CAR-T cells co-expressing the iCasp suicide gene in the presence of 50 U / ml IL-2, either without further treatment or in the presence of 100 nM dasatinib, and the iCaspase-inducing drug 10 nM AP20187 Was cultured in the absence and presence of. After 24 hours, cells were labeled with anti-CD3 mAB and the presence of iCasp + T cells was analyzed by flow cytometry.

Preparation of dasatinib The lyophilized dasatinib was purchased from Selleck Chemicals (Houston, TX, USA) and reconstituted in DMSO (AppliChem, Darmstadt, Germany) to give a stock solution at a concentration of 10 mM. Standard solutions were prepared by DMSO or further dilution in a suitable medium.

Preparation of dexamethasone Dexamethasone (Sigma Aldrich, Steinheim, Germany) was reconstituted in DMSO (AppliChem, Darmstadt, Germany) to obtain a stock solution at a concentration of 100 mM. Standard solutions were prepared by DMSO or further dilution in a suitable medium.

Preparation of Other Tyrosine Kinase Inhibitors Nilotinib, lapatinib and imatinib were purchased from Cell Signaling (Leiden, Netherlands) and reconstituted in DMSO (Sigma Aldrich) to obtain stock solutions at 10 mM concentrations each. Saracatinib, bosutinib and PP1 inhibitors were purchased from Selleck Chemicals (Houston, TX, USA) and reconstituted in DMSO to give stock solutions at 10 mM concentrations each. Standard solutions were prepared by DMSO or further dilution in a suitable medium.

in vivo experiment
All mouse experiments were approved by the UKW Animal Care and Use Committee. NOD.Cg-Prkdc ^scid Il2rg ^tm1Wjl / SzJ (NSG) mice (female, 6-8 weeks old) were purchased from Charles River (Sulzfeld, Germany). Mice were inoculated with 1 × 10 ⁶ Raji / ffluc_GFP tumor cells via tail intravenous injection (iv). Mice were treated with 5 × 10 ⁶ CAR-modified or control non-transduced T cells (CD4: CD8 ratio = 1: 1) via tail intravenous injection (iv). Dasatinib was administered by intraperitoneal injection (ip) at a dose of 10 mg / kg dasatinib (continuous treatment) or 5 mg / kg (intermittent treatment). Tumor loading and distribution were analyzed by continuous bioluminescence imaging on an IVIS Lumina imager (Perkin Elmer, Baesweiler, Germany). Mice were given an ip injection of 0.3 mg / g luciferin, and images were acquired 10 minutes after the injection of luciferin in a small binning mode with an acquisition time of 1 second to 1 minute to obtain unsaturated images. Data were analyzed using LivingImage Software (Caliper) to analyze mean brightness (or photon flux) in the region of interest that included the entire body of each individual mouse. Mice were sacrificed at the end of the experiment and human T cells in bone marrow, peripheral blood and spleen were analyzed by flow cytometry. The presence of (human) cytokines in serum was measured using multiplex cytokines.

(Example 2)
Dasatinib blocks CAR-T cell function
A) Dasatinib blocks the function of CD19 CAR-T cells and ROR1 CAR-T cells Dasatinib blocks the cytolytic activity of CD8 ⁺ CAR-T cells We from n = 3 healthy donors to CD8 + CAR -T cell lines were prepared. In each T cell line, we used EGFRt transduction markers to concentrate CAR-expressing T cells to a purity of> 90%. We used K562, which we transduced with either CD19 (for testing CD19 CAR-T cells) or ROR1 (for testing ROR1 CAR-T cells) as target cells. The cytolytic activity of CD8 ⁺ CAR-T cells was analyzed in a luminescence-based cytotoxicity assay. Dasatinib was added to the assay medium at the start of the assay.

The data show that dasatinib can completely block the cytolytic function of CD8 + T cells expressing CD19 CAR with 4-1BB co-stimulation. The degree of dasatinib-induced blockade of cytolytic CAR-T cell function is dose-dependent (Fig. 2A).
--At a concentration of ≤12.5 nM dasatinib in the assay medium, the cytolytic function of CAR-T cells was not significantly affected (93% specific of target cells by non-dasatinib treated CAR-T cells at t = 12h). Specific lysis of> 88% of target cells by treated CAR-T cells compared to lysis);
--There was partial inhibition of the cytolytic function of CAR-T cells at a concentration of 25 nM dasatinib in the assay medium (compared to 53% specific lysis by non-dasatinib treated CAR-T cells at t = 6h). 26% specific lysis of target cells; and 73% specific lysis of target cells compared to 93% specific lysis of target cells by non-dasatinib-treated CAR-T cells at t = 12h);
--There was (almost) complete inhibition of the cytolytic function of CAR-T cells at a concentration of ≥50 nM dasatinib in assay medium (specific lysis of less than 7% of target cells up to t = 6h; and t = Less than 12% specific lysis of target cells compared to 93% specific lysis of target cells by non-dasatinib-treated CAR-T cells in 12h).

We confirmed that dasatinib can completely block the cytolytic function of CD8 + T cells expressing CD19 CAR, which has a CD28 co-stimulating domain (Fig. 2B).
--At a concentration of 50 nM dasatinib in the assay medium, there was partial inhibition of the cell lytic function of CAR-T cells (at t = 5h, 52% specific lysis of target cells by non-dasatinib treated CAR-T. Specific lysis of less than 23% of target cells compared to specific lysis of 91% of target cells by non-dasatinib-treated CAR-T cells at t = 10h; Melt);
--There was (almost) complete inhibition of the cytolytic function of CAR-T cells at a concentration of 100 nM dasatinib in assay medium (91% specific lysis by non-dasatinib treated CAR-T cells at t = 10h). By comparison, less than 10% specific lysis of target cells at any given time point).

We also confirmed that dasatinib can completely block the cytolytic function of CD8 + T cells expressing ROR1 CAR with a 4-1BB costimulatory domain (Fig. 2C).
--At a concentration of 25 nM dasatinib in the assay medium, there is less than 2% specific lysis of target cells compared to 73% specific lysis by non-dasatinib treated CAR-T cells up to t = 5h, and Less than 2% of target cells at any given time point, compared to 94% specific lysis by non-dasatinib treated CAR-T cells at t = 10h at a concentration of ≥50 nM dasatinib in the assay medium. There was specific dissolution.

Dasatinib present inventors to block the production and secretion of cytokines in CD8 ⁺ CAR-T cells were analyzed for production and secretion of cytokines in the presence or absence of dasatinib CD8 ⁺ CAR-T cell line. CAR-T cells were co-cultured with K562 transduced with either CD19 (for testing CD19 CAR-T cells) or ROR1 (for testing ROR1 CAR-T cells) by the inventors. Dasatinib was added to the assay medium at the start of the co-culture assay. ELISA was performed to detect IFN-γ and IL-2 in the supernatant removed from the co-culture.

The data show that dasatinib can completely block cytokine production and secretion in CD8 + T cells expressing CD19 CAR with 4-1BB co-stimulation. The degree of dasatinib-induced blockade of cytokine production and secretion is dose-dependent (Fig. 3A):
—— At a concentration of ≧ 6.25 nM dasatinib in assay medium, less than 45% residual specific IFN-γ production and less than 60% residual specific IL-2 compared to non-dasatinib treated CAR-T cells. There was production;
--At a concentration of ≥50 nM dasatinib in the assay medium, there was no residual specific IFN-γ production and less than 1% residual specific IL-2 production compared to non-dasatinib treated CAR-T cells. It was.

We have confirmed that dasatinib can completely block the production and secretion of cytokines in CD8 + T cells expressing CD19 CAR, which has a CD28 co-stimulating domain (Fig. 3B).
--With a residual specific IFN-γ production of less than 4.5% compared to non-dasatinib-treated CAR-T cells at a concentration of ≥50 nM dasatinib in assay medium, and no residual specific IL-2 production. It was.

We also confirmed that dasatinib can completely block cytokine production and secretion in CD8 + T cells expressing ROR1 CAR with a 4-1BB costimulatory domain (Fig. 3C).
--At a concentration of ≥50 nM dasatinib in the assay medium, there was less than 3% residual IFN-γ and less than 8.5% residual Il-2 production compared to non-dasatinib treated CAR-T cells.

These data are evidence of the fact that dasatinib is a suitable inhibitor of cytokine secretion by CAR-T cells independently of receptor design and specificity.

Dasatinib present inventors that blocks the proliferation of CD8 ⁺ CAR-T cells was analyzed growth in the presence or absence of dasatinib CD8 ⁺ CAR-T cell line. CAR-T cells were labeled with CFSE and co-invented with K562 in which we transduced either CD19 (for testing CD19 CAR-T cells) or ROR1 (for testing ROR1 CAR-T cells). It was cultured. Dasatinib was added to the assay medium at the start of the co-culture assay. Flow cytometric analysis was performed to determine T cell proliferation at the end of the co-culture assay. A proliferation index, which indicates the average number of cell divisions that occurred during the assay period, was calculated and used to normalize the proliferation index of CAR-T cells stimulated in the absence of dasatinib as 100%. Proliferation was determined.

The data show that dasatinib can completely block the proliferation of CD8 + T cells expressing CD19 CAR with 4-1BB co-stimulation. The degree of dasatinib-induced blockade of proliferation is dose-dependent (Fig. 4A):
--There is less than 80% residual growth compared to non-dasatinib-treated CAR-T cells at a concentration of ≥3.125 nM dasatinib in assay medium;
--There is less than 45% residual growth compared to non-dasatinib-treated CAR-T cells at a concentration of ≥12.5 nM dasatinib in assay medium;
--There was less than 8% residual proliferation compared to non-dasatinib-treated CAR-T cells at a concentration of ≥50 nM dasatinib in assay medium.

We have confirmed that dasatinib can completely block the proliferation of CD8 + T cells expressing CD19 CAR, which has a CD28 co-stimulating domain (Fig. 4B).
--There was less than 7% residual proliferation compared to non-dasatinib-treated CAR-T cells at a concentration of ≥50 nM dasatinib in the assay medium.

We also confirmed that dasatinib can completely block the proliferation of CD8 + T cells expressing ROR1 CAR with a 4-1BB costimulatory domain (Fig. 4C).
--There was less than 7% residual proliferation compared to non-dasatinib-treated CAR-T cells at a concentration of ≥50 nM dasatinib in the assay medium. Stimulation with IL-2 was used as a positive control and reference.

Dasatinib present inventors to block the production and secretion of cytokines in CD4 ⁺ CAR-T cells were analyzed for production and secretion of cytokines in the presence or absence of dasatinib CD4 ⁺ CAR-T cell line. CAR-T cells were co-cultured with K562, which we transduced with CD19. Dasatinib was added to the assay medium at the start of the co-culture assay. Multiplex cytokine analysis was performed in the supernatant removed from the co-culture.

The data show that dasatinib can completely block the production and secretion of cytokines in CD4 + T cells expressing CD19 CAR with 4-1BB co-stimulation. The degree of dasatinib-induced blockade of cytokine production and secretion is dose-dependent (Fig. 5A):
--At a concentration of ≥25 nM dasatinib in the co-culture assay medium, the production and secretion of GM-CSF, IFN-γ, IL-2, IL-4, IL-5, IL-6 and IL-8 is non-dasatinib. > 95% of (almost) completely blocked (for GM-CSF, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-8) compared to treated CAR-T cells Reduction).

We have confirmed that dasatinib can completely block the production and secretion of cytokines in CD4 + T cells expressing CD19 CAR, which has a CD28 co-stimulating domain (Fig. 5B).
--At a concentration of ≥25 nM dasatinib in the co-culture assay medium, the production and secretion of GM-CSF, IFN-γ, IL-2, IL-4, IL-5, IL-6 and IL-8 is non-dasatinib. > 95% of (almost) completely blocked (for GM-CSF, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-8) compared to treated CAR-T cells Reduction).

B) Dasatinib blocks the function of SLAMF7 CAR-T cells Dasatinib blocks the cytolytic activity of CD8 ⁺ SLAMF7 CAR-T cells We from n = 2 healthy donors to SLAMF7-specific CD8 ⁺ CAR- A T cell line was prepared. In each T cell line, we used EGFRt transduction markers to concentrate CAR-expressing T cells to a purity of> 90%. We analyzed the cytolytic activity of CD8 ⁺ CAR-T cells in a bioluminescence-based cytotoxicity assay using SLAMF7 transduced K562 as a target cell. K562-SLAMF7 was produced by the lentiviral transduction of the full-length human SLAMF7 gene. Dasatinib was added to the assay medium at the start of the assay.

The data show that dasatinib can completely block the cytolytic function of CD8 ⁺ T cells expressing SLAMF7-CAR with 4-1BB co-stimulation. The degree of dasatinib-induced blockade of cytolytic CAR-T cell function is dose-dependent (Figure 6A, Chart above; see also Figure 1D for the structure of SLAMF7-CAR with 4-1BB co-stimulation):
--There was partial inhibition of the cytolytic function of CAR-T cells at a concentration of 20 nM dasatinib in the assay medium (specific lysis of 63% of target cells by non-dasatinib treated CAR-T cells at t = 6h). 21% specific lysis of target cells compared to, and 35% specific lysis of target cells compared to 83% specific lysis of target cells by non-dasatinib-treated CAR-T cells at t = 12h) ;
--There was (almost) complete inhibition of the cytolytic function of CAR-T cells at a concentration of ≥40 nM dasatinib in assay medium (specific lysis of less than 5.5% of target cells up to t = 6h; and t Less than 10% specific lysis of target cells compared to 83% specific lysis of target cells by non-dasatinib-treated CAR-T cells at = 12h).

We also confirmed that dasatinib can completely block the cytolytic function of CD8 ⁺ T cells expressing SLAMF7 CAR, which has a CD28 co-stimulation domain (Fig. 6A, lower panel; has a CD28 co-stimulation domain). See also Figure 1E for the structure of SLAMF7 CAR).
--There was partial inhibition of the cytolytic function of CAR-T cells at concentrations of 20, 40 and 60 nM dasatinib in the assay medium (67% of target cells by non-dasatinib treated CAR-T at t = 6h). Less than 21% specific lysis of target cells compared to specific lysis; and less than 35% of target cells compared to 85% specific lysis of target cells by non-dasatinib-treated CAR-T cells at t = 12h Specific dissolution of the residue);
--There was (almost) complete inhibition of the cytolytic function of CAR-T cells at a concentration of ≥80 nM dasatinib in assay medium (85% specific lysis by non-dasatinib treated CAR-T cells at t = 12h). Specific lysis of less than 3% of target cells at any given time point compared to).

Dasatinib blocks cytokine production and secretion in CD8 ⁺ and CD4 ⁺ SLAMF7 CAR-T cells We prepared SLAMF7-specific CD8 ⁺ and CD4 ⁺ CAR-T cell lines from healthy donors with n = 2. .. In each T cell line, we used EGFRt transduction markers to concentrate CAR-expressing T cells to a purity of> 90%. We analyzed the production and secretion of cytokines in CD8 ⁺ and CD4 ⁺ CAR-T cell lines in the presence or absence of dasatinib. CAR-T cells were co-cultured with K562 transduced with SLAMF7 by the present inventors. Dasatinib was added to the assay medium at the start of the co-culture assay. ELISA was performed to detect IFN-γ and IL-2 in the supernatant removed from the co-culture.

The data show that dasatinib can completely block the production and secretion of cytokines in CD8 + T cells expressing SLAMF7 CAR with 4-1BB co-stimulation. The degree of dasatinib-induced blockade of cytokine production and secretion is dose-dependent (Fig. 6B):
--At a concentration of 20 nM dasatinib in the assay medium, there was less than 15% residual specific IFN-γ production compared to non-dasatinib treated CAR-T cells, and no residual specific IL-2 production. ;
--With a residual specific IFN-γ production of less than 0.8% compared to non-dasatinib treated CAR-T cells at a concentration of ≥40 nM dasatinib in the assay medium, and no residual specific IL-2 production. It was.

We have confirmed that dasatinib can completely block the production and secretion of cytokines in CD8 + T cells expressing SLAMF7 CAR with the CD28 co-stimulating domain (Fig. 6B).
--With a residual specific IFN-γ production of less than 4% compared to non-dasatinib-treated CAR-T cells at a concentration of ≥20 nM dasatinib in assay medium, and no residual specific IL-2 production. It was.
--With a concentration of ≥40 nM dasatinib in the assay medium, less than 0.2% residual specific IFN-γ production compared to non-dasatinib treated CAR-T cells, and no residual specific IL-2 production. It was.

We also confirmed that dasatinib can completely block the production and secretion of cytokines in CD4 ⁺ T cells expressing SLAMF7 CAR with a 4-1BB costimulatory domain (Fig. 6C).
--At a concentration of ≥20 nM dasatinib in the assay medium, there was less than 0.5% residual IFN-γ compared to non-dasatinib treated CAR-T cells, and no residual Il-2 production.

We also confirmed that dasatinib can completely block cytokine production and secretion in CD4 ⁺ T cells expressing SLAMF7 CAR with the CD28 co-stimulating domain (Fig. 6C).
--At a concentration of ≥20 nM dasatinib in the assay medium, there was less than 1.2% residual IFN-γ and no residual Il-2 production compared to non-dasatinib treated CAR-T cells.

These data provide additional evidence of the fact that dasatinib is a preferred inhibitor of cytokine secretion by CAR-T cells independently of receptor design and specificity.

C) Dasatinib blocks CAR-T cell signaling Dasatinib blocks the phosphorylation of kinases involved in CAR signaling We have 4-1BB in the presence or absence of 100 nM dasatinib. CD8 ⁺ T cells expressing stimulated CD19 CAR were co-cultured with RCH-ACV target cells (CD19 +) and Western blot analysis was performed to determine the kinase phosphorylation status presumed to be involved in CAR signaling. ..

Phosphorylation of Lck / Src family kinase tyrosine 416, CAR CD3 zeta tyrosine 142, and ZAP70 tyrosine 319 in CAR-T cells co-cultured by us in the presence of dasatinib was performed by us. It was lower compared to CAR-T cells co-cultured in the absence of dasatinib (Fig. 7A). For reference and control, we performed ancillary Western blots for Lck, CAR CD3 zeta, and ZAP70, and β-actin in both dasatinib-treated and untreated CAR-T cells. .. The CD3 zeta domain (CAR CD3 zeta) contained in CAR was distinguished from the endogenous CD3 zeta by its distinct molecular weight.

Quantitative Western blot analysis showed that phosphorylation in dasatinib-treated CAR-T cells was 12.86% (CAR CD3 zeta), 21.57% (Lck) and 21.57% (Lck), respectively, compared to CAR-T cells co-cultured in the absence of dasatinib. It was shown to be only 11.61% (ZAP70) (Fig. 7B).

Dasatinib blocks NFAT-mediated induction of GFP expression CD8 ⁺ CD19 CAR / 4-1BB T cells transduced by us to co-express the NFAT-induced GFP reporter gene Was prepared. We co-culture these T cells with Raji (CD19 +) or K562 (CD19) tumor cells in either the presence or absence of 100 nM dasatinib and perform flow cytometric analysis to perform GFP. The expression of the reporter gene was determined.

The data show that in the presence of dasatinib, induction of GFP reporter gene expression was completely inhibited. The average fluorescence intensity (MFI) of GFP expression in the absence of dasatinib was 1211 on average after stimulation with Raji and only 129 after stimulation with Raji in the presence of dasatinib. It is similar to the background MFI (MFI 117) obtained using unstimulated T cells (Fig. 8, left panel).

We confirmed that the presence of dasatinib interferes with NFAT signaling and GFP reporter gene expression in CD4 ⁺ CD19 CAR / 4-1BB T cells (Fig. 8, right panel).

The data show that dasatinib completely blocks CAR signaling in both CD8 ⁺ T cells and CD4 ⁺ T cells and prevents the expression of NFAT transcription factors.

Interference with signal transduction by dasatinib does not reduce CAR-T cell viability We have CD8 ⁺ CD19 CAR / 4-1BB T cells alone, either in the absence or in the presence of 100 nM dasatinib. It was cultured for 24 hours in or as a co-culture with irradiated K562 / CD19. At the end of co-culture, we stained with anexin-V and 7-AAD to produce live CAR-T cells (anexin-V negative / 7-AAD negative), apoptotic CAR- Percentages of T cells (annexin-V positive / 7-AAD negative) and dead CAR-T cells (anexin-V positive / 7-AAD positive) were determined.

Data show a higher proportion of live CAR-T cells and in the presence of dasatinib after stimulation with K562 / CD19 tumor cells and in the absence of dasatinib (live: 25.4%; apoptosis: 66.2%; death 8.4%). It shows that there was a lower percentage of dead or apoptotic CAR-T cells (live: 47.4%; apoptosis: 45.7%; death 6.9%) (Fig. 9). Similar effects were observed when dasatinib was added to the co-cultures of CAR-T cells and K562 / CD19 tumor cells 2 hours after the start of the co-culture assay (live: 41.7%; apoptosis: 51.3%; death 7). %). These data indicate that dasatinib can protect CAR-T cells from activation-induced cell death (AICD) after encounter with tumor cells.

Taken together, the data show that dasatinib can completely block the stimulation, activation and subsequent effector function of dormant CAR-T cells. Blocking is effective in both CD8 + T cells and CD4 + T cells and functions independently of antigen specificity and the specific design of the CAR construct (eg, the co-stimulatory moiety). Blockade of CAR-T cell function by dasatinib is dose-dependent. Partial inhibition of CAR-T cell function can also be achieved, which depends on the selected concentration of dasatinib.

(Example 3)
Dasatinib blocks the function of activated CAR-T cells Dasatinib blocks the function of activated CAR-T cells CAR-T in which dasatinib is already activated and is performing an effector function Attempts have been made to determine if cell function can be blocked. We prepared CD8 ⁺ CAR-T cell lines from healthy donors with n = 3. In each T cell line, we used EGFRt transduction markers to concentrate CAR-expressing T cells to a purity of> 90%. We analyzed the cytolytic activity of CD8 ⁺ CAR-T cells in a bioluminescent-based cytotoxicity assay using K562 target cells transduced with CD19 by us. Dasatinib was added to assay medium 1 hour after the start of co-culture (dasa + 1h). For comparison, we included a setting in which dasatinib was added at the very beginning of co-culture (dasa; as was done in the experiment in Example 2) and a setting in which dasatinib was not added (untreated).

The data show that dasatinib can block the cytolytic function of already activated CD8 ⁺ T cells expressing CD19 CAR with 4-1BB co-stimulation. In a setting where dasatinib (100 nM) was added to the assay medium 1 hour after the start of co-culture, we detected a reduction in the percentage of target cells specifically lysed up to 7 hours of co-culture. After 7 hours, the percentage of specifically lysed target cells reached a plateau and did not increase further, with 34% specific lysis at t = 10 hours (Fig. 10A). For comparison, there was a much faster and more stable increase in specific target cell lysis over the entire 10 hour assay period in the setting where dasatinib was not added to the co-culture. At each analysis time point above 2 hours, the percentage of specifically lysed target cells was higher compared to the setting with delayed (+1 hour) dasatinib addition. At 10 hours of analysis, the percentage of specifically lysed target cells was> 90% (Fig. 10A). In the setting where dasatinib (100 nM) was added at the start of co-culture, there was a complete blockade of cell lytic activity, consistent with the data obtained in Example 2.

We also show that dasatinib can block the production and secretion of cytokines in already activated CD8 ⁺ T cells that express CD19 CAR with 4-1BB co-stimulation. CD8 ⁺ CAR-T cells were co-cultured with K562 / CD19 target cells for 20 hours, and the presence of IFN-γ and IL-2 in the supernatants obtained from these co-cultures was analyzed by ELISA. The data show lower levels of IFN-γ and IL-2 in the setting with dasatinib (100 nM) added to the assay medium 2 hours after the start of co-culture compared to the setting without dasatinib (untreated control). It is shown that it was (Fig. 10B). After normalization (level of cytokine production in untreated CAR-T cells = 100%), the percentages of residual IFN-γ and Il-2 production were 51% and 28%, respectively (Fig. 10B).

We also show that dasatinib can block the proliferation of already activated CD8 ⁺ T cells expressing CD19 CAR with 4-1BB co-stimulation. CD8 ⁺ CAR-T cells were labeled with CFSE and co-cultured with K562 / CD19 target cells. CAR-T cell proliferation was analyzed after 72 hours based on CFSE dye dilution and a proliferation index was calculated. Dasatinib (100 nM) was added either at the start of co-culture (0 h), or 1 hour (+ 1 h), 3 hours (+ 3 h), or 48 hours (+ 48 h) after the start of co-culture. It was. The proliferation observed in CAR-T cells stimulated with K562 / CD19 target cells in the absence of dasatinib was used as a reference (proliferation = 100%). The data show that the addition of dasatinib 1 hour and 3 hours after the start of co-culture led to lower growth indices of 26% and less than 72% compared to untreated CAR-T cells (Figure). 10C). Addition of dasatinib 48 hours after the start of co-culture led to a lower growth index of 91% compared to untreated CAR-T cells, but this difference was not statistically significant (Fig. 10C). ..

Taken together, these data indicate that dasatinib can block the function of CAR-T cells that are already activated and performing effector functions. This ability has specific clinical relevance to reduce toxicity or prevent exacerbation of toxicity in the context of CAR-T cell immunotherapy.

(Example 4)
Dasatinib Prevents CAR-T Cell Activation During Sequential Stimulation We investigated the effect of dasatinib on activated CAR-T cells with respect to signaling levels using the NFAT / GFP reporter system, and We evaluated whether dasatinib-mediated inhibition could be persistent over time and during successive antigen encounters (Figure 11). NFAT reporter CAR T cells were generated as described in Example 1.

We analyzed the NFAT-promoting expression of GFP in CD8 ⁺ and CD4 ⁺ CAR-T after co-culture with the K562 target cells transduced with CD19 by the present inventors. Dasatinib was added to the assay medium 1 hour after the start of co-culture (dasa + 1h) or during the start of the assay (dasa). For comparison, we included a setting (untreated) without the addition of dasatinib. Subsequent addition of target cells and 100 nM dasatinib simultaneously every 24 hours.

The data show that when dasatinib was added 1 hour after the start of the assay, T cells were partially activated and showed reduced GFP expression on day 1 (untreated but stimulated). 734 MFIs compared to 1949 in CAR-T cells). When dasatinib was present from the beginning, GFP expression was completely suppressed on day 1 when dasatinib was present from the beginning (MFI 137).

The data show that once dasatinib is present in both T cells treated from the beginning and T cells treated 1 hour after the start of the assay, GFP is induced by subsequent stimulation on day 2 or 3. Indicates that it was not. Instead, GFP levels decreased until day 3, indicating that further antigen-specific stimulation was prevented by dasatinib and cells remained functionally off.

We confirmed that dasatinib prevents subsequent antigen-specific stimulation in CD19 CAR co-expressing the 4-1BB co-stimulation domain and the NFAT / GFP reporter system and CD4 ⁺ T cells (Fig. 11). ).

The data show that when dasatinib was added 1 hour after the start of the assay, T cells were partially activated and showed reduced GFP expression on day 1 (untreated but stimulated). 841 MFI compared to 2288 in CAR-T cells). When dasatinib was present from the beginning, GFP expression was completely suppressed on day 1 (MFI 317).

The data show that once dasatinib is present in both T cells treated from the beginning and T cells treated 1 hour after the start of the assay, GFP is induced by subsequent stimulation on day 2 or 3. Indicates that it was not. Instead, GFP levels decreased until day 3 (MFI 108 and MFI 326, respectively), but GFP expression remained high in untreated CAR-T cells (MFI 2499), with additional antigen-specific stimulation dasatinib. Prevented by, indicating that the cells were kept functionally off.

Taken together, these data support that dasatinib can block the function of already activated CAR-T cells. Furthermore, the data show that dasatinib can interfere with already induced activation and prevent subsequent induction of transcription factors in spite of the presence of antigen.

(Example 5)
Blockade of CAR-T cell function is rapid and completely reversible after removal of dasatinib
Blockade of CAR-T cell function is rapid and completely reversible after short-term exposure to dasatinib We present CD8 ⁺ T expressing CD19 CAR with 4-1BB co-stimulation from healthy donors with n = 3. A cell line was prepared. In each T cell line, we used EGFRt transduction markers to concentrate CAR-expressing T cells to a purity of> 90%. We analyzed the cytolytic activity of CD8 ⁺ CAR-T cells in a bioluminescent-based cytotoxicity assay using K562 target cells transduced with CD19 by us.

Dasatinib (100 nM) was added to assay medium at the start of co-culture (t = -2h). After 2 hours (t = 0h), the assay medium was discarded and replaced with fresh assay medium (ie, dasatinib was removed). The cytolytic activity of CAR-T cells was analyzed at 1-hour intervals for 10 hours. For comparison, we included a setting in which dasatinib was not present in the assay medium (Fig. 12A).

The data were consistent with the data obtained in Example 2 in the presence of dasatinib (ie, in the first 2 hours of the co-culture assay), and CAR-T cells did not exhibit any cytolytic activity. Is shown. However, immediately after the medium change (ie, immediately after the removal of dasatinib), CAR-T cells began to exert cytolytic activity. At +4 hours, CAR-T cells conferred 77% specific lysis of target cells. At the end of the co-culture assay at 10 hours, CAR-T cells were> 95% specific to the target cells, similar to CAR-T cells that were not treated with dasatinib during the first 2 hours of co-culture. The target lysis was imparted (Fig. 12A). The data were supported using CD8 ⁺ T cell lines expressing CD19 CAR with CD28 co-stimulation prepared by us from healthy donors with n = 2 (Fig. 12B).

Long-term exposure to dasatinib does not reduce CAR-T cell viability 50 U / ml for 8 consecutive days, either in the absence of dasatinib [(-)] or in the presence of dasatinib [100 nM, (+)] CD8 ⁺ CD19 CAR / 4-1BB-T cells were maintained in the culture medium supplemented with IL-2. Dasatinib was added to the culture medium every 24 hours.

On day 2 (ie, short-term exposure of 48 hours) and day 8 (long-term exposure), we obtained an aliquot of CAR-T cells from each culture condition and used staining with 7AAD and Anexin V. The cell viability was determined. At each time point, the percentage of viable CAR-T cells was higher in CAR-T cell lines maintained in the presence of dasatinib when compared to CAR-T cells cultured without dasatinib (Fig. 13). The data show that both short-term and long-term exposure to dasatinib do not lead to decreased survival of CAR-T cells.

Blockade of CAR-T cell function is rapid and completely reversible after exposure to dasatinib and subsequent removal 50 U / ml for 7 consecutive days, either in the absence of dasatinib or in the presence of dasatinib (100 nM) CD8 ⁺ CD19 CAR / 4-1BB-T cells were maintained in the culture medium supplemented with IL-2. Dasatinib was added to the culture medium every 24 hours.

After 1 day (ie, short-term exposure for 24 hours) and 7 days (long-term exposure), we obtained an aliquot of CAR-T cells from each culture condition and made a complete medium change to remove dasatinib. did. Next, we performed functional tests to assess whether previous exposure to dasatinib affected the subsequent ability of CAR-T cells to exert antitumor function. The data show that after both short-term and long-term exposure to dasatinib and subsequent removal of dasatinib, CAR-T cells are antitumor at the same level and potency as CAR-T cells cultured in the absence of dasatinib. Indicates that the function was exhibited.

The data in Figure 14A show that after 1 day (left chart) and 7 days (right chart) exposure to dasatinib, and subsequent removal of dasatinib, CAR-T cells were not exposed to dasatinib at all. It is shown that the target cells exhibited rapid and strong specific cytolytic activity comparable to that of T cells (no dasatin / no dasatin compared to no dasatin). The data in Figure 14A also show after 1-day (left chart) and 7-day (right chart) exposure to dasatinib, subsequent removal (washing) of dasatinib, and new exposure to dasatinib (dasa / dasa). It is shown that the complete blockade of the cytolytic activity of CAR-T cells was still functioning.

The data in FIG. 14B show that after 1 and 7 days of exposure to dasatinib, and subsequent removal of dasatinib, CAR-T cells were comparable to CAR-T cells (pre-Dasa) that were not exposed to dasatinib at all. , Shows that IFN-γ (left chart) and IL-2 (right chart) were produced and secreted in response to stimulation with target cells. The data in Figure 14B also show complete blockade of cytokine production and secretion by CAR-T cells after 1 and 7 days of exposure to dasatinib, subsequent removal of dasatinib and new exposure to dasatinib (between Dasa). Indicates that was still working.

The data in FIG. 14C show that after 1 and 7 days of exposure to dasatinib and subsequent removal of dasatinib, CAR-T cells were comparable to CAR-T cells (pre-Dasa) that were not exposed to dasatinib at all. It shows that it proliferated in response to stimulation with target cells. The data in Figure 14C also show that complete blockade of CAR-T cell proliferation still functions after 1-day and 7-day exposure to dasatinib, subsequent removal of dasatinib, and new exposure to dasatinib (between Dasa). Show that it was.

Taken together, these data show that blockade of CAR-T cell function by dasatinib does not negatively affect CAR-T cell viability, and that blockade of CAR-T cell function is independent of pretreatment duration. It is shown to be rapidly and completely reversible after removal of dasatinib. Previous exposure to dasatinib does not preclude dasatinib's ability to block CAR-T cell function in repeated exposures. These data indicate that dasatinib can be used to precisely and highly effectively control the function of CAR-T cells.

(Example 6)
Dasatinib blocks CAR-T cell function in vivo and prevents cytokine release syndrome Dasatinib blocks CAR-T cell function in mouse xenograft lymphoma models We are immunodeficient mice (NSG / Raji) The effect of dasatinib on CD19 CAR / 4-1BB-T cells was evaluated in vivo using a xenograft model. Experimental settings and treatment schedules are provided in Figure 15A. Briefly, a cohort of n ≧ 3 mice was inoculated with 1 × 10 ^ 6 firefly-luciferase_GFP transduced Raji tumor cells on day 0 and the mice were CAR transduced T cells or on day 7. Treated with any of the control non-transduced T cells. The T cell product consisted of equal proportions of CD4 ⁺ T cells and CD8 ⁺ T cells (1: 1 ratio), with a total dose of 5 × 10 ^ 6 T cells. In each treatment cohort, dasatinib was given to a subgroup of mice starting 3 hours before T cell transfer, followed by a total of 6 doses every 6 hours.

Based on the known pharmacokinetics and pharmacodynamics of dasatinib in mice [23] (assuming that the pharmacokinetics after ip injection is not faster than after iv injection), this is because dasatinib is at least 100 nM in mouse serum. Windows from 3 hours before T cell transfer present to 33 hours after T cell transfer (total window: 36 hours) were provided. In this mouse model, blockade with dasatinib should therefore be effective up to +1 day of CAR-T cell transfer and no longer effective up to +3 day of CAR-T cell transfer.

Dasatinib blocks the production and secretion of cytokines in CAR-T cells and prevents cytokine release in vivo We have treated mice with CAR-T cells and dasatinib in parallel (NSG / Raji). Serum cytokine levels were analyzed in. To determine cytokine levels, we performed multiplex cytokine analysis in mouse sera (Fig. 15B).

The data show GM-T cells and dasatinib-fed mice (+1 day, CAR / +) compared to CAR-T cells-fed and no dasatinib-fed mice (CAR /-). CSF (6.4 pg / ml), IFN-γ (13.4 pg / ml), TNF-α (0.04 pg / ml), IL-2 (less than the detection limit), IL-5 (21.4 pg / ml) and IL-6 It shows that there were significantly lower serum levels (less than detected) [ie, 3.2% GM-CSF, 1.7% IFN-γ, 0.3% TNF-α and 2.6% IL-5 levels] (Figure). 15B). The data support our previous in vitro observation (see Example 2) that dasatinib can block the cytokine secretion of CAR-T cells. The data also support our previous in vitro observation (see Example 5) that the blockade of CAR-T cell function by dasatinib is rapidly reversible (Fig. 15B).

On day +3 of the experiment in which dasatinib was discontinued, cytokine serum levels were 45.8 pg / ml compared to the serum cytokine levels observed on day +1 (dosetinib was being administered) in the same mouse. GM-CSF, 411.8 pg / ml IFN-γ, 0.9 pg / ml TNF-α, 0.2 pg / ml IL-2, 331.2 pg / ml IL-5 and 0.9 pg / ml IL-6 [ It increased to 7.2 for GM-CSF, 30.7 for IFN-γ, 22.9 for TNF-α, and a multiple of 15.5 for IL-5 secretion] (Fig. 15B).

Taken together, these data indicate that i) cytokine production and secretion in CAR-T cells can be blocked by dasatinib in vivo; ii) cytokine production and secretion blockade by repeated administration of dasatinib at least 36 It can be maintained for a long time; iii) Blocking of cytokine secretion is shown to be reversible after discontinuation of dasatinib in vivo.

CAR-T cell function is blocked in vivo in the presence of dasatinib, and CAR-T cells resume antitumor function in vivo when exposure to dasatinib is discontinued. The antitumor function of CAR-T cells was analyzed in mice fed either non-chimericated control T cells and given dasatinib or no dasatinib according to the treatment schedule in FIG. 15A. Raji tumor loading was determined by bioluminescence imaging on days 1, 1 and 3.

Data show that between day 1 and day 1, mice fed CAR-T cells + dasatinib (CAR / +; multiples of 14.1) were non-transduced control T cells and mice fed dasatinib. (Control / +; multiple change of 15.4) and similar rate of tumor progression (Fig. 15C, black bar), indicating that CAR was ineffective. For comparison, tumor progression was significantly slower at this short interval in mice fed CAR-T cells without dasatinib.

The data showed a strong reduction in tumor load in both groups treated with CAR-T cells (previously with or without dasatinib) between days +1 to +3 when dasatinib was discontinued. This indicates that there was a strong reduction in tumor load, especially in mice previously treated with dasatinib, explaining that the blockade of CAR-T function by dasatinib was rapidly reversible in vivo (). Figure 15C, gray bar).

We used flow cytometry to analyze the presence of human T cells in mouse bone marrow (BM), spleen (SP) and peripheral blood (PB) at d1 and d3 after T cell injection. .. Viable human T cells, 7AAD ^-, were identified as CD3 ⁺ and CD4 ^+.

Data show that on day 1, the frequency of human T cells was CAR-T compared to mice fed CAR-T cells and not dasatinib (CAR / untreated: BM: 0.099%; PB 0.16%). No difference in cells and dasatinib-fed mice (CAR / treatment: BM: 0.087%; PB: 0.19%), that is, administration of dasatinib did not impair CAR-T cell engraftment (Fig. 15D). , D + 1). On day +3, the frequency of CAR-T cells was parallel treated with dasatinib compared to mice not fed dasatinib (CAR / untreated: BM: 0.56%; PB: 0.61%). Lower at (CAR / Treatment: BM: 0.23%; PB: 0.36%) (Fig. 15D, d + 3), dasatinib can block the proliferation and expansion of CAR-T cells in vitro. It was consistent with the observation of.

Dasatinib blocks CAR signaling and induction of NFAT transcription factors in CAR-T cells in vivo CD8 transduced by us to co-express the NFAT-induced GFP reporter gene ^{+ And} CD4 ⁺ CD19CAR / 41BB T cells were prepared. Using a xenograft mouse model as described above (Fig. 15A), we performed flow cytometric analysis to perform during (d + 1) or after (d + 3) dasatinib treatment. Expression of the GFP reporter gene was determined in human T cells isolated from the bone marrow and spleen of mice treated with either CAR-T cells or control T cells in the presence or absence of dasatinib.

The data show that in the presence of dasatinib, expression of the GFP reporter gene in CAR-T cells obtained from bone marrow and spleen was significant in mice treated with dasatinib compared to mice not treated with dasatinib. Shows that it was lower (Fig. 15E). The mean fluorescence intensity (MFI) of GFP in bone marrow CAR-T cells was 10687 in the absence of dasatinib (CAR / untreated) at d + 1 and only 6967 in the presence of dasatinib (CAR / treatment). , This is a 35% reduction. A similar reduction in GFP reporter gene expression was observed in d + 1 splenic CAR-T cells (reduction: 36%). At d + 3, where dasatinib was no longer effective, the difference between previously treated CAR-T cells in the bone marrow and untreated CAR-T cells was only 10% and was previously treated in the spleen. Only 23% between CAR-T cells and untreated CAR-T cells.

Taken together, these data indicate that dasatinib can control CAR-T cell function in vivo. In particular, the data show that administration of dasatinib prevents cytokine release from CAR-T cells and prevents cytokine release syndrome. Treatment with dasatinib does not impair T cell engraftment. When exposure to dasatinib is discontinued, CAR-T cells resume antitumor function.

(Example 7)
Dasatinib is activated in vivo Blocks the function of CD19 CAR / 4-1BB CAR-T cells Dasatinib blocks CAR-T cell function in mouse xenograft lymphoma models We are immunodeficient mice (NSG / Raji) ), The effect of dasatinib on activated CAR / 4-1BB-T cells was evaluated in vivo using a xenograft model. The experimental settings and treatment schedule are provided in Figure 16A. Briefly, a cohort of mice with n ≧ 6 was inoculated with 1 × 10 ^ 6 firefly-luciferase_GFP transduced Raji tumor cells on day 0 and the mice were CAR transduced T cells or on day 7. Treated with any of the control non-transduced T cells. The T cell product consisted of equal proportions of CD4 ⁺ T cells and CD8 ⁺ T cells (1: 1 ratio), with a total dose of 5 × 10 ^ 6 T cells. In the indicated cohort, mice were given dasatinib at a total of 8 doses 3 days after T cell transfer and every 6 hours thereafter. Based on the known pharmacokinetics and pharmacodynamics of dasatinib in mice, this is because dasatinib is present in mouse serum at concentrations above the threshold required to block the function of CAR-T cells 10 after tumor inoculation. Provided windows from day 12 to day 12. CAR-T cell function is off in the presence of dasatinib and is reignited to function on when dasatinib administration is discontinued.

We have anti-CAR-T cells in mice fed either CAR-T cells or non-transduced control T cells and were fed or not fed dasatinib according to the treatment schedule in FIG. 16A. Tumor function was analyzed. Raji tumor loading was determined by bioluminescence imaging on days 7, 10, 12, 14, 17, and once weekly thereafter (Fig. 16B). Data show that in the first phase (7th-10th days) after T cell transfer, CD19-CAR T cells begin to exert antilymphoma activity and delay lymphoma progression, as demonstrated by BLI. It shows that it was made (Fig. 16B). In the second phase (10th to 12th days) after T cell transfer, dasatinib rapidly induces a function-off state and an anti-lymphoma response, as evidenced by a strong increase in BLI signaling in the dasatinib-treated cohort. Stopped sex. In contrast, BLI signaling was not increased during this period in mice fed CD19-CAR T cells but not dasatinib. In the third phase (after day 12), dasatinib was discontinued to allow CAR T cells to return to their function-on state. CAR T cells rapidly resumed anti-lymphoma function, as evidenced by a rapid decrease in BLI signal. After day 17, CAR-T cells were more effective in controlling tumors in the cohort treated with dasatinib, and tumors were controlled up to day 59 in all mice. In contrast, tumors recurred in the majority of mice in the cohort fed CAR-T cells but not dasatinib (Fig. 16B).

Data show that when compared to control T cell-fed mice (2018% growth rate) on days 7-10, CAR-T cell-fed mice were CAR-fed mice and subsequently. Shows that they showed a reduction in tumor growth comparable to that in mice determined to be fed dasatinib (growth rates of 298% and 227%, respectively). On days 10-12, mice fed CAR-T cells + dasatinib (CAR (on / off / on); 405%) were mice fed CAR-T cells alone (CAR (on)). It showed a much faster rate of tumor progression than 22.2%) (Fig. 16C), ie CAR was ineffective in the presence of dasatinib despite primary activation of CAR-T cells. Data show a strong reduction in tumor load in both groups treated with CAR-T cells on days 12-17 when dasatinib was discontinued (previous dasatinib with or without; 66% and 97.8, respectively). % Reduction of tumor luminescence), especially in mice previously treated with dasatinib, indicating a strong reduction in tumor loading, with dasatinib blocking CAR-T function rapidly and reversibly in vivo. Explain that (Fig. 16C).

Dasatinib blocks the production and secretion of cytokines from CAR-T cells in vivo to prevent cytokine release syndrome We have treated mice with CAR-T cells and dasatinib in parallel (NSG / Serum cytokine levels were analyzed in Raji). To assess cytokine expression, we performed an analysis of IFNγ in mouse serum (Fig. 16D).

The data show that in mice fed CAR-T cells, IFNγ serum levels were equal on day 10, ie prior to dasatinib administration. On day 12, after dasatinib administration, mice fed dasatinib (CAR (on / off /) compared to mice fed CAR-T cells and not dasatinib (CAR (on)) (157 pg / ml) There was a significantly lower serum level (24 pg / ml) of IFN-γ in (on)) (Fig. 16D). The data show that dasatinib can block cytokine secretion from activated CAR-T cells and prevent subsequent stimulation of inhibited T cells by our previous in vitro observations (implemented). See Example 3 and Example 4).

The data also support our previous in vitro observations (see Example 5) that the blockade of CAR-T cell function by dasatinib is rapidly reversible. Cytokine serum levels increased to 38 pg / ml IFN-γ on day 14 of the experiment in which dasatinib was discontinued, which was observed in the same mice on day 12 (dose dasatinib was administered). It is a multiple of 1.6 change compared to cytokine levels (Fig. 16D).

Taken together, these data indicate that i) cytokine production and secretion in activated CAR-T cells can be blocked by dasatinib in vivo; ii) cytokine production and secretion can be blocked by at least repeated administration of dasatinib. Can be maintained for 54 hours; iii) Blocking of cytokine secretion is shown to be reversible after discontinuation of dasatinib in vivo.

(Example 8)
Dasatinib is activated in vivo Blocks the function of CD19 / CD28 CAR-T cells Dasatinib blocks CAR-T cell function in a mouse xenograft lymphoma model We in immunodeficient mice (NSG / Raji) A xenograft model was used to assess the effect of dasatinib on activated CAR / CD28-T cells in vivo. Experimental schedule settings and treatment schedules are provided in Figure 17A. Briefly, a cohort of mice with n ≧ 8 was inoculated with 1 × 10 ^ 6 firefly-luciferase_GFP transduced Raji tumor cells on day 0 and the mice were CAR transduced T cells or on day 7. Treated with any of the control non-transduced T cells. The T cell product consisted of equal proportions of CD4 ⁺ T cells and CD8 ⁺ T cells (1: 1 ratio), with a total dose of 5 × 10 ^ 6 T cells. In the indicated cohort, mice were given dasatinib at a total of 8 doses every 6 hours 3 days after T cell transfer, ie 10 days. Based on the known pharmacokinetics and pharmacodynamics of dasatinib in mice, this is because dasatinib is present in mouse serum at concentrations above the threshold required to block the function of CAR-T cells 10 after tumor inoculation. Provided windows from day 12 to day 12. As a control, a cohort of mice fed CAR-T cells was additionally treated with a dasatinib-free vehicle (pointed as CAR / DMSO).

CAR-T cell function is off in the presence of dasatinib and is reignited to function on when dasatinib administration is discontinued.

We have anti-CAR-T cells in mice fed either CAR-T cells or non-transduced control T cells and were fed or not fed dasatinib according to the treatment schedule in FIG. 17A. Tumor function was analyzed. Raji tumor loading was determined by bioluminescence imaging on days 7, 10, 12, 14, 17, and once weekly thereafter. Data show that CD19-CAR T cells begin to exert antilymphoma activity and are strongly activated during the first phase (7th-10th days) after T cell transfer, as evidenced by a decrease in BLI. Show that. At the same time, tumors grew rapidly in mice fed CAR T cells and dasatinib (Fig. 17B). In the second phase (10th-12th days) after T cell transfer, dasatinib rapidly induces a function-off state, arrests anti-lymphoma responsiveness, and tumors occur in 10 animals in the dasatinib-treated cohort. Seven of them started regrowth. In contrast, BLI signals were given in 9 of 10 mice that received CD19-CAR T cells but no additional treatment, as well as CD19-CAR T cells and dasatinib-free vehicles. In 8 of the 10 mice, there was a rapid reduction during this period. In the third phase (after day 12), dasatinib was discontinued to allow CAR T cells to return to their function-on state. In fact, CAR T cells rapidly resumed anti-lymphoma function (Fig. 17B), giving CAR-T cells and vehicles or CAR-T cells alone, as evidenced by a rapid decrease in BLI signal. Day 17 led to greater remission (507 median BLI) when compared to mice (966 and 839 median BLI, respectively).

Data show that CAR-T cell-fed mice showed reduced tumor growth when compared to control T-cell-fed mice (1628% growth rate) on days 7-10, T cells. Pointed to activation (75% (dasa), 15% (DMSO) and 11% (CAR only) tumor reduction, respectively) (Fig. 17C). From day 10 to day 12, mice fed CAR-T cells + dasatinib (CAR (on / off / on)); 33% growth) were fed CAR-T cells alone (32%). Tumor progression (Fig. 17C), such as (CAR / DMSO) and 61% (CAR /-) reduction in BLI), ie CAR is in the presence of dasatinib despite primary activation of CAR-T cells. Was not effective.

Data show a strong reduction in tumor load in all groups treated with CAR-T cells on days 12-14 when dasatinib was discontinued (9 of 10 in CAR / DMSO, And in the CAR alone cohort 6/10), there was a strong reduction in tumor load, especially in mice previously treated with dasatinib (10 out of 10 mice, an average reduction in BLI of 71%). Explain that the blockade of CAR-T function by dasatinib was rapidly reversible in vivo (Fig. 17C).

(Example 9)
Dasatinib exerts superior control over CAR-T cell function compared to dexamethasone We have selected CD8 + CD19 CAR-T cell lines with a 4-1BB costimulatory domain from healthy donors with n = 3. Prepared. In each T cell line, we used EGFRt transduction markers to concentrate CAR-expressing T cells to a purity of> 90%. We evaluated the effect of dexamethasone on CAR-T cell function by conducting functional tests using K562 transduced with CD19 as a target cell by the present inventors. Dexamethasone was added to the assay medium at the start of the assay or used for 24-hour pretreatment at the indicated dose.

Dasatinib exerts superior control over the cytolytic function of CAR-T cells compared to dexamethasone We analyzed the cytolytic activity of CD8 + CAR-T cells in a bioluminescent-based cytotoxicity assay. did. The data show that dexamethasone cannot completely block the cytolytic function of CD8 + CAR-T cells expressing CD19 CAR with a 4-1BB costimulatory domain. The degree of dexamethasone-induced inhibition of cell lysis function was largely dose-independent, but rather treatment schedule-dependent:
--When dexamethasone was added to the assay medium at the start of the assay, the cytolytic function of CAR-T cells was not significantly affected at any dose applied (Fig. 18A, left panel) (untreated CAR-T). Specific lysis of target cells> 87% by treated CAR-T cells compared to 91% specific lysis of cell-mediated target cells).
--When CAR-T cells were pretreated with dexamethasone for 24 hours (Fig. 18A, right panel), there was only partial inhibition of the cytolytic function of CAR-T cells at all tested doses (untreated CAR). -> 45% specific lysis of target cells by dexamethasone-treated CAR-T cells at t = 10h compared to 91% specific lysis of target cells mediated by T cells).
--Complete blockade of specific lysis of target cells mediated by CAR-T cells was observed for cells treated with 0.1 μM dasatinib at the start of the assay, which cells are both for comparison as a reference. Included in the panel (<1% specific dissolution at t = 10h).

Dasatinib exerts superior control over the production and secretion of cytokines by CAR-T cells compared to dexamethasone We have cytokines from CD8 ⁺ CAR-T cell lines in the presence or absence of dexamethasone. Production and secretion of. ELISA was performed to detect IFN-γ and IL-2 in the supernatant removed from the co-culture.

The data show that dexamethasone cannot completely block cytokine secretion in CD8 + CAR-T cells expressing CD19 CAR with a 4-1BB costimulatory domain. The effect of dexamethasone on cytokine secretion depends on the treatment schedule and differs for different cytokines:
--There was no significant reduction in IFN-γ secretion for CAR-T cells pretreated (black bar) or treated only during the assay (gray bar) (Fig. 18B, left panel). At any given concentration of dexamethasone, there was more than 43% of residual specific IFN-γ secretion by CAR-T cells treated with dexamethasone compared to untreated CAR-T cells.
--There was a partial reduction in IL-2 secretion for CAR-T cells treated with dexamethasone (gray bar) only during pretreatment (black bars) or assay (Figure 18B, right panel). At any given concentration of dexamethasone, there was less than 17% residual specific IL-2 secretion by CAR-T cells treated with dexamethasone compared to untreated CAR-T cells. CAR-T cells treated with 0.1 μM dasatinib showed complete blockade of IL-2 secretion, which was consistent with the data obtained in Experiment 2 and included for comparison as a reference.

Dasatinib exerts superior control over CAR-T cell proliferation compared to dexamethasone We analyzed the proliferation of CD8 ⁺ CAR-T cell lines in the presence or absence of dexamethasone. CAR-T cells were labeled with CFSE and co-cultured with K562 transduced with CD19 by the present inventors. Flow cytometric analysis was performed to determine T cell proliferation after 72 hours. A proliferation index, which indicates the average number of cell divisions that occurred during the assay period, was calculated and used to normalize the proliferation index of CAR-T cells stimulated in the absence of further treatment as 100%. Residual proliferation was determined.

The data support that dexamethasone can reduce the proliferation of CD8 ⁺ CAR-T cells. The effect was equal between CAR-T cells given 24h pretreatment with dexamethasone and CAR-T cells given dexamethasone at the start of co-culture. At any given concentration, residual proliferation was less than 26% compared to CAR-T cells left untreated (Fig. 18C). Nonetheless, complete blockade (<5.6%) of CAR-T cell proliferation as observed with 0.1 μM dasatinib could not be achieved by dexamethasone.

Taken together, these data indicate that dasatinib exerts superior control over CAR-T cells compared to dexamethasone. In particular, the data show that administration of dexamethasone to CAR-T cells is observed by treatment of CAR-T cells with 0.1 μM dasatinib, both at the start of co-culture and 24 hours before the assay. -Indicates that complete blockade of T cell function cannot be achieved.

(Example 10)
Tyrosine kinase inhibitors can affect CAR-T cell effector function
Effect of dasatinib and other clinically approved tyrosine kinase inhibitors on CAR-T cell function We have CD8 ⁺ ROR1 CAR-T with a 4-1BB co-stimulation domain from healthy donors with n = 2. A cell line was prepared. In each T cell line, we used EGFRt transduction markers to concentrate CAR-expressing T cells to a purity of> 90%. We performed functional tests using ROR1 ⁺ RCH-ACV as target cells to evaluate the effect of clinically approved TKI panels on CAR-T cell function. TKIs were added to assay medium at the start of the assay to final concentrations of 100 nM dasatinib, 5.3 μM imatinib, 4.2 μM lapatinib or 3.6 μM nilotinib. Untreated CAR-T cells were used as controls for the calculation.

We analyzed the cytolytic activity of CD8 ⁺ CAR-T cells in a bioluminescent-based cytotoxicity assay. The data show that dasatinib is the only TKI in the panel tested that can completely block cell lysis function (specific lysis <5% at t = 8 hours) (Fig. 19A). The data indicate that there was partial inhibition of the cytolytic function of CAR-T cells in the presence of lapatinib, nilotinib or imatinib (> 90 mediated by untreated CAR-T cells at t = 8 hours). <75% specific lysis mediated by CAR-T cells treated with either lapatinib, nilotinib or imatinib compared to% specific lysis).

The present inventors can produce and secrete IFN-γ in CD8 ⁺ CAR-T cells by performing ELISA using the supernatant removed from the co-culture of CAR-T cells with RCH-ACV target cells. Analyzed. The data show that dasatinib and nilotinib can reduce the amount of IFN-γ production and secretion (Fig. 19B):
-In the presence of 100 nM dasatinib, the production and secretion of IFN-γ was completely blocked and below detection levels.
--In the presence of 3.6 μM nilotinib, IFN-γ production and secretion was reduced to 480 pg / ml compared to 1310 pg / ml produced by untreated CAR-T cells, which is a residual of 36.6%. Similar to IFNγ secretion.

We then analyzed the proliferation of CD8 ⁺ CAR-T cell lines in the presence or absence of TKI treatment. CAR-T cells were labeled with CFSE and co-cultured with RCH-ACV. CAR-T cell proliferation was evaluated by flow cytometry 72 hours after co-culture.

The data show that treatment with 100 nM dasatinib mediates (almost) complete inhibition of CAR-T cell proliferation, similar to the data shown in Example 2. The data also show that nilotinib can partially block the growth of CD8 ⁺ CAR-T cells, compared to untreated CAR-T cells with a growth index of 2.84 at a concentration of 3.6 μM nilotinib in the assay medium. The proliferation index was reduced to 2.24.

Effects of dasatinib and other Src kinase inhibitors on the cytolytic function of CAR-T cells We prepared a CD8 ⁺ CD19 CAR-T cell line with a 4-1BB co-stimulation domain from a healthy donor. .. In T cell lines, we used EGFRt transduction markers to concentrate CAR-expressing T cells to a purity of> 90%. We analyzed the cytolytic activity of CD8 ⁺ CAR-T cells in a 4-hour bioluminescence-based cytotoxicity assay using CD19-transduced K562 as a target cell. , The effect of the panel of Src kinase inhibitors on the cytolytic function of CAR-T cells was evaluated. Src kinase inhibitors were added to assay medium at the start of the assay over a 4-log concentration range.

The data show that of the four Src kinase inhibitors tested, three can block the cytolytic activity of CAR-T cells (Fig. 20):
--At a concentration of 10 nM dasatinib in the assay medium, there was partial inhibition of the cytolytic function of CAR-T cells (targeted compared to 82% specific lysis of target cells by untreated CAR-T cells). Specific lysis of 16.1% of cells).
--There was (almost) complete inhibition of the cytolytic function of CAR-T cells at a concentration of ≥100 nM dasatinib in the assay medium (compared to the specific lysis of 82% of target cells by untreated CAR-T cells). And <3% specific lysis of target cells).
--There was partial inhibition of the cytolytic function of CAR-T cells at a concentration of 10 nM PP1 inhibitor in assay medium (compared to 82% specific lysis of target cells by untreated CAR-T cells). Specific lysis of 62.4% of target cells).
--There was (almost) complete inhibition of the cytolytic function of CAR-T cells at a concentration of ≥100 nM PP1 inhibitor in assay medium (specific lysis of 82% of target cells by untreated CAR-T cells). <3% specific lysis of target cells compared to).
--There was (almost) complete inhibition of the cytolytic function of CAR-T cells at a concentration of 1000 nM bostinib in assay medium (compared to the specific lysis of 82% of target cells by untreated CAR-T cells). (Specific lysis of <3%) of target cells).

Taken together, these data show that tyrosine kinase inhibitors other than dasatinib can exert inhibitory effects on CAR-T cell function. In particular, the data show that nilotinib is a potent inhibitor of cytokine production and secretion from CAR-T cells. Src kinase inhibitors PP1 inhibitors and bosutinibs can completely block the cytolytic function of CD8 + CAR-T cells.

(Example 11)
Intermittent treatment with dasatinib increases CAR-T cell function Intermittent exposure to dasatinib increases the antitumor function of CAR-T cells in vivo We have immunodeficient mice (NSG / The effect of dasatinib on CD19 CAR / 4-1BB-T cells was evaluated in vivo using a xenograft model in Raji). The experimental settings and treatment schedule are provided in Figure 21A. Briefly, a cohort of mice with n ≧ 2 was inoculated with 1 × 10 ^ 6 firefly-luciferase_GFP transduced Raji tumor cells on day 0. CAR-T cells (ie, CD8 ⁺ and CD4 ⁺ T cells expressing CD19 CAR with a 4-1BB co-stimulation domain, total dose: 5 × 10e6; CD8: CD4 ratio = 1: 1) or control non-transduced T Cells were administered by iv tail intravenous injection on day 7. Dasatinib of 5 mg / kg was administered by ip injection from d7 to d11 every 24 hours, and then by ip injection at d12 and d14 every 36 hours (7 doses in total). Tumor loading was determined on days 7, 9 and 15 by continuous bioluminescence imaging.

Based on the known pharmacokinetics and pharmacodynamics of dasatinib in mice [23], this should lead to a temporary blockade of CAR-T cell function by dasatinib as shown in Example 2> 50 nM. A window was provided approximately 6 hours after each injection present in mouse serum at concentration. Over the next 21 hours (until the next injection), dasatinib should be below the inhibition threshold of 50 nM and therefore should have no inhibitory effect on CAR-T cell function.

The data show that intermittent treatment of mice with dasatinib increases the antitumor function of CAR-T cells in vivo (Fig. 21B). Mice fed CAR-T cells and dasatinib showed better tumor control and slower tumor progression compared to mice fed CAR-T cells without dasatinib. On day 8, the mean bioluminescent signal in mice fed CAR-T cells without dasatinib was 1.9e10p / s / cm * 2 / sr, while intermittent treatment with CAR-T cells and dasatinib was performed. The mean bioluminescence signal was only 5.6e9p / s / cm * 2 / sr in the fed mice (p <0.05). On day 8, there was no statistically significant difference in bioluminescence signal between mice fed non-transduced control T cells with dasatinib and mice fed non-transduced control T cells without dasatinib.

Intermittent exposure to dasatinib increases CAR-T cell engraftment, proliferation and persistence in vivo We use a xenograft model as described in Figure 21A to dasatinib. CAR-T cell engraftment, proliferation and persistence were analyzed in mice with intermittent exposure. On day 15, mice were sacrificed and peripheral blood (PB), bone marrow (BM) and spleen (SP) were analyzed for the presence of human CAR-T cells by flow cytometry. Viable human T cells ^{(7AAD -, CD3 +, CD45} +) is shown in FIG. 22A gating strategy that was used to evaluate the percentage of and residual tumor cells (GFP ^+).

The data show that intermittent exposure to dasatinib increases the antitumor function of CAR-T cells, as shown in Figure 21B. A high tumor burden of 58.8% of all living cells, GFP-positive tumor cells, was detected in the bone marrow of one individual mouse treated with CAR-T cells (Fig. 22A, top panel). In contrast, one exemplary mouse treated with CAR-T cells and intermittent dasatinib showed a residual tumor load of 0.22% of all living cells in the bone marrow (Figure). 22A, lower panel).

The data in FIG. 22B show that intermittent treatment with dasatinib increases CAR-T cell engraftment, proliferation and persistence in vivo. In bone marrow and spleen, the percentage of human CAR-T cells is intermittent when compared to animals (BM: 1.9%, SP: 3.2%) that received CAR-T cells but not intermittent dasatinib. It was higher in animals treated with dasatinib (BM: 7.3%; SP: 6.9%) (p> 0.05).

Taken together, these data show that intermittent exposure to dasatinib increases the antitumor function of CAR-T cells in vivo, and intermittent exposure to dasatinib also increases the life of CAR-T cells in vivo. Shows increased onset, proliferation and persistence.

(Example 12)
Intermittent treatment with dasatinib reduces PD-1 expression on CAR-T cells Based on the mouse model introduced in Example 11 (Fig. 21A), we found bone marrow (BM), peripheral blood. The surface expression of PD-1 on human CAR-T cells in (PB) and spleen (SP) was analyzed by flow cytometry.

The data show that intermittent exposure to dasatinib results in PD-1 expression in bone marrow and peripheral blood CAR-T cells compared to CAR-T cells in the corresponding organs of mice not exposed to intermittent dasatinib. It is shown to be significantly reduced (Fig. 23):
--In bone marrow (BM), the average fluorescence intensity (MFI) obtained after staining CAR-T cells with anti-PD1 mAb was 9461 in mice not exposed to dasatinib (CAR /-), dasatinib. Only 7025 in mice (CAR / +) intermittently treated with.
--In peripheral blood (PB), the average fluorescence intensity (MFI) obtained after staining CAR-T cells with anti-PD1 mAb was 4110 in mice not exposed to dasatinib (CAR /-). It was only 2775 in mice (CAR / +) intermittently treated with dasatinib.
--In the spleen (SP), the mean fluorescence intensity (MFI) obtained after staining CAR-T cells with anti-PD1 mAb was 4318 in mice not exposed to dasatinib (CAR /-), dasatinib. Only 23652 in mice (CAR / +) intermittently treated with.

Taken together, these data indicate that dasatinib reduces PD-1 expression on CAR-T cells upon intermittent exposure.

(Example 13)
CAR-T cells blocked by dasatinib are susceptible to subsequent removal using the iCasp9 suicide gene
The iCasp suicide gene in either the absence or presence of 100 nM dasatinib and in the absence or presence of the iCaspase-inducing drug 10 nM AP20187 in a medium supplemented with 50 U / ml IL-2. CAR-T cells co-expressing the above were cultured. After 24 hours, cells were labeled with anti-CD3 mAB and flow cytometry was used to analyze the presence of iCasp ⁺ T cells.

The data show that induction of suicide genes and subsequent apoptosis of T cells are not affected by dasatinib (Fig. 24):
In the presence of the dimerizing agent (dasatinib / dimerizing agent +), the percentage of iCasp ⁺ cells was reduced to 45%, which is 100 nM dasatinib (36%) and the dimerizing agent (dasatinib +). It was equivalent to the percentage of iCasp ^{+ in} the presence of / dimerizing agent +).

Taken together, these data indicate that CAR-T cells blocked by dasatinib are susceptible to subsequent removal using the iCasp9 suicide gene.

In addition to the immune cells and tyrosine kinase inhibitors for use according to the invention, the materials used for the methods of the invention are claimed according to known standards for the manufacture of pharmaceutical and diagnostic products. It can be industrially produced and marketed as a product for methods and uses (eg, for treating cancer as defined herein). Therefore, the present invention is industrially applicable.

(Reference)

Sequence The amino acid sequence below is part of the construct "4-1BB CD19 CAR with a co-stimulating domain" (see Figure 1A):
SEQ ID NO: 1 (GMCSF signal peptide):
MLLLVTSLLLCELPHPAFLLIP
SEQ ID NO: 2 (CD19 Heavy Chain Variable Domain (VH)):
DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSRYYQESGPGLVAPSQSLSVTCTVSGV
SEQ ID NO: 3 (IgG4 hinge domain):
ESKYGPPCPPCP
SEQ ID NO: 4 (CD28 transmembrane domain):
MFWVLVVVGGVLACYSLLVTVAFIIFWV
SEQ ID NO: 5 (4-1BB costimulatory domain):
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
SEQ ID NO: 6 (CD3z signaling domain):
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
SEQ ID NO: 7 (T2A ribosome skipping sequence):
LEGGGEGRGSLLTCGDVEENPGPR
SEQ ID NO: 8 (GMCSF signal peptide):
MLLLVTSLLLCELPHPAFLLIP
SEQ ID NO: 9 (EGFRt):
RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM

The following amino acid sequence is part of the construct "CD19 CAR with CD28 co-stimulating domain" (see Figure 1B):
SEQ ID NO: 10 (GMCSF signal peptide):
MLLLVTSLLLCELPHPAFLLIP
SEQ ID NO: 11 (CD19 scFv):
DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSYYTFGLVAPSQSLSVTCTVSGVS
SEQ ID NO: 12 (IgG4 hinge domain):
ESKYGPPCPPCP
SEQ ID NO: 13 (CD28 transmembrane domain):
MFWVLVVVGGVLACYSLLVTVAFIIFWV
SEQ ID NO: 14 (CD28 co-stimulation domain):
RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
SEQ ID NO: 15 (CD3z signaling domain):
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
SEQ ID NO: 16 (T2A ribosome skipping sequence):
LEGGGEGRGSLLTCGDVEENPGPR
SEQ ID NO: 17 (GMCSF signal peptide):
MLLLVTSLLLCELPHPAFLLIP
SEQ ID NO: 18 (EGFRt):
RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM

The following amino acid sequence is part of the construct "4-1 BB ROR1 CAR with co-stimulation domain" (see Figure 1C):
SEQ ID NO: 19 (GMCSF signal peptide):
MLLLVTSLLLCELPHPAFLLIP
SEQ ID NO: 20 (hR12 Heavy Chain Variable Domain (VH)):
QVQLVESGGALVQPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEWIATIYPSSGKTYYAASVQGRFTISADNAKNTVYLQMNSLTAADTATYFCARDSYADDGALFNIWGQGTLVTVSS
SEQ ID NO: 21 (4 (GS) x3 linker):
GGGGSGGGGSGGGGS
SEQ ID NO: 22 (hR12 light chain variable domain (VL)):
QLVLTQSPSVSAALGSSAKITCTLSSAHKTDTIDWYQQLAGQAPRYLMYVQSDGSYEKRSGVPDRFSGSSSGADRYLIISSVQADDEADYYCGADYIGGYVFGGGTQLTVG
SEQ ID NO: 23 (IgG4 hinge domain):
ESKYGPPCPPCP
SEQ ID NO: 24 (CD28 transmembrane domain):
MFWVLVVVGGVLACYSLLVTVAFIIFWV
SEQ ID NO: 25 (4-1BB co-stimulation domain):
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
SEQ ID NO: 26 (CD3z signaling domain):
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
SEQ ID NO: 27 (T2A ribosome skipping sequence):
LEGGGEGRGSLLTCGDVEENPGPR
SEQ ID NO: 28 (GMCSF signal peptide):
MLLLVTSLLLCELPHPAFLLIP
SEQ ID NO: 29 (EGFRt):
RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM

The following amino acid sequence is part of the construct "SLAMF7 CAR with 4-1BB costimulatory domain" (see Figure 1D):
SEQ ID NO: 30 (GMCSF signal peptide):
MLLLVTSLLLCELPHPAFLLIP
SEQ ID NO: 31 (huLuc63 Heavy Chain Variable Domain (VH)):
EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYAPSLKDKFIISRDNAKNSLYLQMNSLRAEDTAVYYCARPDGNYWYFDVWGQGTLVTVSS
SEQ ID NO: 32 (4 (GS) x3 linker):
GGGGSGGGGSGGGGS
SEQ ID NO: 33 (huLuc63 light chain variable domain (VL)):
DIQMTQSPSSLSASVGDRVTITCKASQDVGIAVAWYQQKPGKVPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQPEDVATYYCQQYSSYPYTFGQGTKVEIK
SEQ ID NO: 34 (IgG4 hinge domain):
ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGF
SEQ ID NO: 35 (CD28 transmembrane domain):
MFWVLVVVGGVLACYSLLVTVAFIIFWV
SEQ ID NO: 36 (4-1BB co-stimulation domain):
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
SEQ ID NO: 37 (CD3z signaling domain):
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
SEQ ID NO: 38 (T2A ribosome skipping sequence):
LEGGGEGRGSLLTCGDVEENPGPR
SEQ ID NO: 39 (GMCSF signal peptide):
MLLLVTSLLLCELPHPAFLLIP
SEQ ID NO: 40 (EGFRt):
RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM

The following amino acid sequence is part of the construct "SLAMF7 CAR with CD28 co-stimulating domain" (see Figure 1E):
SEQ ID NO: 41 (GMCSF signal peptide):
MLLLVTSLLLCELPHPAFLLIP
SEQ ID NO: 42 (huLuc63 Heavy Chain Variable Domain (VH)):
EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYAPSLKDKFIISRDNAKNSLYLQMNSLRAEDTAVYYCARPDGNYWYFDVWGQGTLVTVSS
SEQ ID NO: 43 (4 (GS) x3 linker):
GGGGSGGGGSGGGGS
SEQ ID NO: 44 (huLuc63 light chain variable domain (VL)):
DIQMTQSPSSLSASVGDRVTITCKASQDVGIAVAWYQQKPGKVPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQPEDVATYYCQQYSSYPYTFGQGTKVEIK
SEQ ID NO: 45 (IgG4 hinge domain):
ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGF
SEQ ID NO: 46 (CD28 transmembrane domain):
MFWVLVVVGGVLACYSLLVTVAFIIFWV
SEQ ID NO: 47 (CD28 co-stimulation domain):
RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
SEQ ID NO: 48 (CD3z signaling domain):
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
SEQ ID NO: 49 (T2A ribosome skipping sequence):
LEGGGEGRGSLLTCGDVEENPGPR
SEQ ID NO: 50 (GMCSF signal peptide):
MLLLVTSLLLCELPHPAFLLIP
SEQ ID NO: 51 (EGFRt):
RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM

Claims (148)

A composition for use in a method of treating cancer in a patient, wherein the composition comprises a tyrosine kinase inhibitor.
In the method, the composition is administered to the patient, and the method is a method of treating cancer, including immunotherapy.
Composition. The composition of claim 1 for use as defined in claim 1, wherein the immunotherapy is adoption immunotherapy. The composition according to claim 1 or 2, wherein the immunotherapy is an immunotherapy using immune cells, for use as defined in claim 1 or 2. The composition according to claim 3, wherein the immunotherapy is an immunotherapy using immune cells expressing a chimeric antigen receptor, for use as defined in claim 3. The composition according to claim 4, wherein the chimeric antigen receptor can bind to an antigen for use as defined in claim 4. The composition according to claim 5, wherein the chimeric antigen receptor can bind to a cell surface antigen for use as defined in claim 5. The composition according to claim 5 or 6, wherein the antigen is a cancer antigen for use as defined in claim 5 or 6. The antigens are CD4, CD5, CD10, CD19, CD20, CD22, CD27, CD30, CD33, CD38, CD44v6, CD52, CD64, CD70, CD72, CD123, CD135, CD138, CD220, CD269, CD319, ROR1, ROR2, SLAMF7, BCMA, αvβ3 integrin, α4β1 integrin, LILRB4, EpCAM-1, MUC-1, MUC-16, L1-CAM, c-kit, NKG2D, NKG2D ligand, PD-L1, PD-L2, Lewis-Y, CAIX , CEA, c-MET, EGFR, EGFRvIII, ErbB2, Her2, FAP, FR-a, EphA2, GD2, GD3, GPC3, IL-13Ra, Mesoterin, PSMA, PSCA, VEGFR, and FLT3. , The composition according to any one of claims 5 to 7 for use as specified in any one of claims 5 to 7. The composition of claim 8 for use as defined in claim 8, wherein the antigen is selected from the group consisting of CD19, CD20, CD22, CD123, SLAMF7, ROR1, BCMA, and FLT3. The composition of claim 9, wherein the antigen is CD19, for use as defined in claim 9. The composition of claim 9, wherein the antigen is ROR1 for use as defined in claim 9. The composition of claim 9, wherein the antigen is BCMA, for use as defined in claim 9. The composition of claim 9, wherein the antigen is FLT3, for use as defined in claim 9. The composition of claim 9, wherein the antigen is CD20, for use as defined in claim 9. The composition of claim 9, wherein the antigen is CD22, for use as defined in claim 9. The composition of claim 9, wherein the antigen is CD123, for use as defined in claim 9. The composition of claim 9, wherein the antigen is SLAMF7, for use as defined in claim 9. The composition according to any one of claims 5 to 17, wherein the cancer comprises cancer cells expressing the antigen, for use as defined in any one of claims 5 to 17. Any one of claims 4-18, wherein the chimeric antigen receptor comprises a co-stimulation domain selected from the group consisting of CD27, CD28, 4-1BB, ICOS, DAP10, NKG2D, MyD88 and OX40 co-stimulation domains. The composition according to any one of claims 4 to 18 for use as specified in. The composition of claim 19 for use as defined in claim 19, wherein the chimeric antigen receptor comprises a CD28 co-stimulating domain. The composition of claim 19, wherein the chimeric antigen receptor comprises a 4-1BB costimulatory domain for use as defined in claim 19. The composition according to claim 19, wherein the chimeric antigen receptor comprises an OX40 co-stimulating domain for use as defined in claim 19. The composition according to any one of claims 3 to 22, wherein the immune cell is a lymphocyte, according to any one of claims 3 to 22. The composition according to any one of claims 3 to 23 for use as defined in any one of claims 3 to 23, wherein the immune cell is a B lymphocyte or a T lymphocyte. The composition of claim 24 for use as defined in claim 24, wherein the immune cell is a T lymphocyte. 25. The composition of claim 25 for use as defined in claim 25, wherein the immune cells are CD4 + and / or CD8 + T lymphocytes. The composition of claim 26 for use as defined in claim 26, wherein the immune cell is a CD4 + T lymphocyte. The composition of claim 26 for use as defined in claim 26, wherein the immune cell is a CD8 + T lymphocyte. The immune cells are CD8 + killer T cells, CD4 + helper T cells, naive T cells, memory T cells, central memory T cells, effector memory T cells, memory stem T cells, invariant T cells, NKT cells, cytokine-induced killer. For use as defined in any one of claims 3-28, selected from the group consisting of T cells, gamma / delta T cells, natural killer cells, monospheres, macrophages, dendritic cells, and granulocytes. The composition according to any one of claims 3 to 28. The composition according to any one of claims 1 to 29, wherein the tyrosine kinase inhibitor is an Src kinase inhibitor, according to any one of claims 1 to 29. The composition according to any one of claims 1 to 30, wherein the tyrosine kinase inhibitor is an inhibitor of a kinase upstream of NFAT, for use as defined in any one of claims 1-30. The composition according to any one of claims 1-31 for use as defined in any one of claims 1-31, wherein the tyrosine kinase inhibitor is an Lck kinase inhibitor. 13. Of claims 1-32, wherein the tyrosine kinase inhibitor is selected from the group consisting of dasatinib, salacatinib, bosutinib, nilotinib, and PP1 inhibitors, for use as defined in any one of claims 1-32. The composition according to any one item. The composition of claim 33 for use as defined in claim 33, wherein the tyrosine kinase inhibitor is dasatinib. The composition of claim 33 for use as defined in claim 33, wherein the tyrosine kinase inhibitor is bosutinib. The composition of claim 33 for use as defined in claim 33, wherein the tyrosine kinase inhibitor is a PP1 inhibitor. The composition of claim 33 for use as defined in claim 33, wherein the tyrosine kinase inhibitor is nilotinib. The composition according to any one of claims 3 to 37 for use as defined in any one of claims 3 to 37, wherein the tyrosine kinase inhibitor causes inhibition of the immune cell. 38. The composition of claim 38 for use as defined in claim 38, wherein the inhibition is inhibition of cell-mediated effector function of the immune cell. The inhibition of the immune cells is their
I) Cell lytic activity and / or
II) Cytokine secretion and / or
III) The composition of claim 38 or 39 for use as defined in claim 38 or 39, which is an inhibition of growth. The composition of any one of claims 38-40 for use as defined in any one of claims 38-40, wherein the inhibition comprises inhibiting PD1 expression in the immune cell. The inhibition of one or more cytokines selected from the group consisting of GM-CSF, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-8, and IL-10. The composition according to any one of claims 38 to 41 for use as defined in any one of claims 38 to 41, comprising inhibiting the cytokine secretion of the immune cells. In any one of claims 38-42 for use as defined in any one of claims 38-42, wherein the inhibition comprises inhibiting IFN-γ and / or IL-2 secretion of the immune cell. The composition described. The composition of claim 43 for use as defined in claim 43, wherein the inhibition comprises inhibiting IFN-γ secretion of the immune cell. The composition of claim 43 for use as defined in claim 43, wherein the inhibition comprises inhibiting IL-2 secretion of the immune cell. The composition according to any one of claims 38 to 45 for use as defined in any one of claims 38 to 45, wherein the inhibition is a partial or complete inhibition. The composition of any one of claims 38-46 for use as defined in any one of claims 38-46, wherein the inhibition does not reduce the viability of the immune cell. The inhibition does not reduce the viability of the immune cells over a given period of time when the composition is administered to the patient, the period being 1 hour, preferably 2 hours, preferably 3 hours, preferably 4. Hours, preferably 5 hours, preferably 6 hours, preferably 8 hours, preferably 12 hours, preferably 18 hours, preferably 1 day, preferably 2 days, more preferably 3 days, even more preferably 7 days, More preferably 2 weeks, even more preferably 3 weeks, even more preferably 4 weeks, even more preferably 2 months, even more preferably 3 months, even more preferably 6 months, claim 47. The composition according to claim 47 for use as specified in. The composition according to any one of claims 38 to 48 for use as defined in any one of claims 38 to 48, wherein the inhibition is reversible. The composition of claim 49 for use as defined in claim 49, wherein the inhibition is reversed after the composition has not been administered to the patient for a given length of time. The given length of time is 3 days, preferably 2 days, more preferably 24 hours, even more preferably 18 hours, even more preferably 12 hours, even more preferably 8 hours, even more preferably 6. The time, more preferably 4 hours, even more preferably 3 hours, even more preferably 2 hours, even more preferably 90 minutes, even more preferably 60 minutes, even more preferably 30 minutes, claim 50. The composition according to claim 50 for the prescribed use. The composition according to any one of claims 1 to 51 for use as specified in any one of claims 1 to 51, wherein the composition is administered continuously or intermittently. The composition of claim 52 for use as defined in claim 52, wherein the composition is administered continuously. The composition of claim 52 for use as defined in claim 52, wherein the composition is administered intermittently. After the initial administration of the composition, the composition is administered such that the serum level of the tyrosine kinase inhibitor is maintained at or above the threshold serum level during the duration of the treatment. The composition according to any one of claims 1 to 54 for use as specified in any one of claims 1 to 54. In the method, the serum level of the tyrosine kinase inhibitor remains above the threshold serum level at least once and below the same threshold serum level at least once during the duration of the treatment after the initial administration of the composition. The composition according to any one of claims 1 to 55 for use as defined in any one of claims 1 to 55, wherein the composition is administered as such. Claim 55 or 56, wherein the threshold serum level is in the range of 0.1 nM to 1 μM, preferably 1 nM to 500 nM, more preferably 5 nM to 100 nM, even more preferably 10 nM to 75 nM, even more preferably 25 nM to 50 nM. The composition according to claim 55 or 56 for use as specified in. 57. The composition of claim 57 for use as defined in claim 57, wherein the threshold serum level is 50 nM. The threshold serum level is the minimum serum level, and at the minimum serum level, the inhibition of the immune cells is their.
I) Cell lytic activity and / or
II) Cytokine secretion and / or
III) The composition of any one of claims 55-58 for use as defined in any one of claims 55-58, which is a complete inhibition of growth. Claims 1-59 for use as defined in any one of claims 1-59, wherein said treatment of cancer has improved clinical outcomes compared to said immunotherapy for said cancer alone. The composition according to any one of the above. Any of claims 1-60 for use as defined in any one of claims 1-60, wherein said use is for reducing or preventing toxicity associated with said immunotherapy for the cancer. The composition according to one item. A claim for use as defined in any one of claims 1-61, wherein said use is for reducing tumor burden in said patient as compared to said immunotherapy for said cancer alone. Item 8. The composition according to any one of Items 1 to 61. The provision of any one of claims 1 to 62, wherein the use in said treatment of cancer does not reduce the therapeutic effect of said immunotherapy for said cancer as compared to said immunotherapy for said cancer alone. The composition according to any one of claims 1 to 62 for use in. Any of claims 1-63, wherein said use in said treatment of cancer is use to increase the therapeutic effect of said immunotherapy for said cancer as compared to said immunotherapy for said cancer alone. The composition according to any one of claims 1 to 63 for use prescribed in one paragraph. Claims 1-34, wherein said use in said treatment of cancer is use to reduce disease and mortality of said immunotherapy for said cancer as compared to said immunotherapy for said cancer alone. The composition according to any one of claims 1 to 64 for use as specified in any one. Claims 1-65, wherein said use in said treatment of cancer is use to increase the antitumor efficacy of said immunotherapy for said cancer as compared to said immunotherapy for said cancer alone. The composition according to any one of claims 1 to 65 for use as specified in any one of the above. The use of the immune cells in the treatment of the cancer alone as compared to the engraftment and / or persistence of the immune cells in the immunotherapy for the cancer / Or the composition according to any one of claims 3-66 for use as specified in any one of claims 3-66, which is a use for increasing persistence. Use to increase the engraftment of said immune cells in said immunotherapy as compared to said use in said treatment of cancer alone in a method comprising said immunotherapy for said cancer. The composition according to any one of claims 3 to 67 for use as specified in any one of claims 3 to 67. Claimed that the use is to reduce the exhaustion of the immune cells in the immunotherapy for the cancer as compared to the exhaustion of the immune cells in a method comprising the immunotherapy for the cancer alone. The composition according to any one of claims 3 to 68 for use as specified in any one of paragraphs 3 to 68. The composition
I) Prior to the treatment of cancer with immunotherapy and / or
II) In parallel with the aforementioned treatment of cancer with immunotherapy and / or
III) The composition according to any one of claims 1 to 69 for use as specified in any one of claims 1 to 69, which is administered after the treatment of cancer by immunotherapy. The composition of claim 70 for use as defined in claim 70, wherein the composition is administered prior to said treatment of cancer by immunotherapy. The composition of claim 70 for use as defined in claim 70, wherein the composition is administered in parallel with said treatment of cancer by immunotherapy. The composition of claim 70 for use as defined in claim 70, wherein the composition is administered after said treatment of cancer by immunotherapy. The composition of claim 70 for use as defined in claim 70, wherein the composition is administered prior to the treatment of cancer by immunotherapy and in parallel with said treatment of cancer by immunotherapy. The composition of claim 70 for use as defined in claim 70, wherein the composition is administered prior to said treatment of cancer by immunotherapy and after said treatment of cancer by immunotherapy. The composition of claim 70 for use as defined in claim 70, wherein the composition is administered in parallel with said treatment of cancer by immunotherapy and after said treatment of cancer by immunotherapy. As defined in claim 70, the composition is administered prior to the treatment of cancer by immunotherapy, in parallel with the treatment of cancer by immunotherapy, and after the treatment of cancer by immunotherapy. The composition according to claim 70 for use. Any one of claims 3 to 77 for use as defined in any one of claims 3 to 77, wherein said use is for preventing activation of said immune cells in said immunotherapy. The composition according to. The composition of claim 78 for use as defined in claim 78, wherein the immune cell is a dormant immune cell. The composition according to any one of claims 3 to 79 for use as defined in any one of claims 3 to 79, wherein the immune cell is of human origin. The composition of claim 80 for use as defined in claim 80, wherein the immune cell of human origin is a primary human cell. The composition of claim 81 for use as defined in claim 81, wherein the primary human cell is a primary human T lymphocyte. The composition of any one of claims 80-82 for use as defined in any one of claims 80-82, wherein the immune cell of human origin is an allogeneic cell with respect to the patient. The composition of any one of claims 80-82 for use as defined in any one of claims 80-82, wherein the immune cell of human origin is a syngeneic cell with respect to the patient. Any one of claims 4 to 84 for use as defined in any one of claims 4 to 84, wherein the immune cell is an immune cell that transiently or stably expresses the chimeric antigen receptor. The composition according to the section. Any one of claims 4-85 for use as defined in any one of claims 4-85, wherein the chimeric antigen receptor is a first, second, or third generation chimeric antigen receptor. The composition according to paragraph 1. The use according to any one of claims 5 to 86, wherein the chimeric antigen receptor comprises a single chain variable fragment, preferably the single chain variable fragment can bind to the antigen. The composition according to any one of 5 to 86. Claims 5-86 for use as defined in any one of claims 5-86, wherein the chimeric antigen receptor comprises a ligand or a fragment thereof and the ligand or fragment thereof can bind to the antigen. The composition according to any one of the above. The chimeric antigen receptor is selected from the group consisting of CD3 zeta, CD3 epsilon, CD3 gamma, T cell receptor alpha chain, T cell receptor beta chain, T cell receptor delta chain, and T cell receptor gamma chain. The composition according to any one of claims 5 to 88 for use as defined in any one of claims 5 to 88, comprising a signaling domain comprising one or more domains. Any one of claims 1-89 for use as defined in any one of claims 1-89, wherein said cancer is a cancer associated with a higher risk of disease and death in said immunotherapy. The composition according to. The cancer comprises cells expressing one or more checkpoint molecules, wherein the one or more checkpoint molecules are preferably A2AR, B7-H3, B7-H4, BTLA, CTLA-4, Claims 1-90 for use as defined in any one of claims 1-90, selected from the group consisting of IDO, KIR, LAG3, PD-L1, PD-L2, TIM-3, and VISTA. The composition according to any one of the above. The composition of claim 91 for use as defined in claim 91, wherein the cancer comprises cells expressing PD-L1. 15. Of claims 1-92 for use as defined in any one of claims 1-92, wherein the cancer is a cancer selected from the group consisting of carcinoma, sarcoma, myeloma, leukemia, and lymphoma. The composition according to any one item. The composition of claim 93 for use as defined in claim 93, wherein the cancer is myeloma. The composition of claim 93 for use as defined in claim 93, wherein the cancer is leukemia. The composition of claim 93 for use as defined in claim 93, wherein the cancer is lymphoma. The cancer for use as defined in claim 93, wherein the cancer is a carcinoma, preferably a carcinoma selected from the group consisting of breast cancer, lung cancer, colorectal cancer, and pancreatic cancer. The composition according to claim 93. The composition of claim 95 for use as defined in claim 95, wherein the leukemia is B-cell leukemia, T-cell leukemia, myelogenous leukemia, acute lymphoblastic leukemia, or chronic myelogenous leukemia. The composition of claim 96 for use as defined in claim 96, wherein the lymphoma is a non-Hodgkin's lymphoma, a Hodgkin's lymphoma, or a B-cell lymphoma. The cancer
I) CD19 positive and / or
II) BCMA positive and / or
III) ROR1 positive and / or
IV) FLT3 positive and / or
V) CD20 positive and / or
VI) CD22 positive and / or
VII) CD123 positive and / or
VIII) The composition of any one of claims 1-99 for use as defined in any one of claims 1-99, which is a cancer characterized as SLAMF7 positive. The composition of claim 100 for use as defined in claim 100, wherein the cancer is CD19 positive. The composition of claim 100 for use as defined in claim 100, wherein the cancer is BCMA positive. The composition of claim 100 for use as defined in claim 100, wherein the cancer is ROR1 positive. The composition of claim 100 for use as defined in claim 100, wherein the cancer is FLT3 positive. The composition of claim 100 for use as defined in claim 100, wherein the cancer is CD20 positive. The composition of claim 100 for use as defined in claim 100, wherein the cancer is CD22 positive. The composition of claim 100 for use as defined in claim 100, wherein the cancer is CD123 positive. The composition of claim 100 for use as defined in claim 100, wherein the cancer is SLAMF7 positive. Any one of claims 1-108 for use as defined in any one of claims 1-108, wherein said patient is a patient who is not eligible for the treatment of the cancer by said immunotherapy alone. The composition according to the section. Claims 1-109 for use as defined in any one of claims 1-109, wherein said patient is not eligible for conventional adoptive immunotherapy using T cells expressing a chimeric antigen receptor. The composition according to any one item. The composition of any one of claims 1-110 for use as defined in any one of claims 1-110, wherein said patient has an increased risk of developing cytokine release syndrome. Any one of claims 1-111 for use as defined in any one of claims 1-111, wherein the patient has an increased risk of developing neurotoxic side effects associated with said immunotherapy. The composition according to. One of claims 1-112 for use as defined in any one of claims 1-112, wherein the patient has an increased risk of developing an on-target / off tumor effect associated with said immunotherapy. The composition according to paragraph 1. For use as defined in any one of claims 1-113, wherein the patient has elevated serum levels of one or more cytokines selected from the group of IFN-γ, IL-6, and MCP1. The composition according to any one of claims 1 to 113. For use as defined in any one of claims 1-114, wherein the patient is a patient who has developed an immune response to the immunotherapy, wherein the immune response is a side effect of the immunotherapy for the cancer. The composition according to any one of claims 1 to 114. The composition according to any one of claims 1 to 115 for use as defined in any one of claims 1 to 115, wherein the treatment method is a treatment method combined with allogeneic or autologous hematopoietic stem cell transplantation. Stuff. The composition according to any one of claims 1 to 116, wherein the composition further comprises a pharmaceutically acceptable carrier, according to any one of claims 1 to 116. The composition according to any one of claims 1 to 117 for use as defined in any one of claims 1 to 117, wherein the composition is administered by a route other than oral administration. The cancer is a cancer other than chronic myelogenous leukemia and acute lymphoblastic leukemia, according to any one of claims 1 to 118 for use prescribed in any one of claims 1 to 118. Composition. A composition for use in a method of treating one or more side effects associated with immunotherapy in a patient, wherein the composition comprises a tyrosine kinase inhibitor.
And, in the method, the composition is administered to the patient.
Composition. The composition according to claim 120 for use according to claim 120, wherein the immunotherapy is the immunotherapy according to any one of claims 2 to 17 and 19 to 29. The composition according to claim 120 or 121 for use according to claim 120 or 121, wherein the cancer is the cancer according to any one of claims 18, 90 to 108, and 119. The composition of claims 120-122 for use as defined in claims 120-122, wherein the patient is the patient as defined in any one of claims 109-115. The composition according to claims 120 to 123 for use as defined in claims 120 to 123, wherein the use is the use specified in any one of claims 1 to 119. The one or more side effects associated with immunotherapy
I) Cytokine release syndrome and / or
II) Macrophage activation syndrome and / or
III) Off-target toxicity and / or
IV) On-target / off tumor recognition of normal and / or malignant cells, and / or
V) Rejection of immunotherapeutic cells and / or
VI) Unintentional activation of immunotherapeutic cells and / or
VII) Tonic signaling and activation of immunotherapeutic cells and / or
VIII) Neurotoxicity and / or
IX) The composition according to any one of claims 120 to 124 for use as defined in any one of claims 120 to 124, selected from the group consisting of tumor lysis syndrome. The composition of claim 125 for use as defined in claim 125, wherein the side effect associated with immunotherapy is cytokine release syndrome. The composition of claim 125 for use as defined in claim 125, wherein the side effect associated with immunotherapy is off-target toxicity. The composition of claim 125 for use as defined in claim 125, wherein the side effect associated with immunotherapy is on-target / off tumor recognition of normal and / or malignant cells. The composition of claim 125 for use as defined in claim 125, wherein the side effect associated with immunotherapy is rejection of immunotherapeutic cells. The composition of claim 125 for use as defined in claim 125, wherein the side effect associated with immunotherapy is unintended activation of immunotherapeutic cells. The composition of claim 125 for use as defined in claim 125, wherein the side effect associated with immunotherapy is tonic signaling and activation of immunotherapeutic cells. The composition of claim 125 for use as defined in claim 125, wherein the side effect associated with immunotherapy is neurotoxicity. The composition of claim 125 for use as defined in claim 125, wherein the side effect associated with immunotherapy is tumor lysis syndrome. One or more of the cytokine release syndromes selected from the group consisting of GM-CSF, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-8, and IL-10. The composition of claim 126 for use as defined in claim 126, characterized by elevated cytokine serum levels of cytokines. The composition of claim 134 for use as defined in claim 134, wherein said use is for causing a reduction in one or more of the elevated cytokine serum levels. The composition of claim 134 or 135 for use as defined in claim 134 or 135, wherein the cytokine release syndrome is caused by the immunotherapy. A composition for use in a method of regulating cells expressing a chimeric antigen receptor in immunotherapy for treating cancer in a patient, wherein the composition comprises a tyrosine kinase inhibitor.
And, in the method, the composition is administered to the patient.
Composition. The composition according to claim 137 for use according to claim 137, wherein the immunotherapy is the immunotherapy according to any one of claims 2 to 17, 19 to 29, and 125 to 136. .. The composition according to claim 137 or 138 for use according to claim 137 or 138, wherein the cancer is the cancer according to any one of claims 18, 90 to 108, and 119. The patient according to any one of claims 109 to 115, according to any one of claims 137 to 139 for use according to any one of claims 137 to 139. Composition. The use according to any one of claims 137 to 140 for use according to any one of claims 137 to 140, wherein the use is the use specified in any one of claims 1 to 136. Composition. I) Immune cells and
II) A composition comprising a tyrosine kinase inhibitor. The composition according to claim 142, wherein the immune cell is the immune cell according to any one of claims 3 to 17, 19 to 29, and 79 to 89. The composition according to claim 142 or 143, wherein the tyrosine kinase inhibitor is the tyrosine kinase inhibitor according to any one of claims 30 to 59. The composition according to any one of claims 142 to 144, wherein the composition comprises a pharmaceutically acceptable carrier. For use as specified in any one of claims 1-141,
I) Immune cells and
II) Combination with tyrosine kinase inhibitor. The combination according to claim 144, wherein the immune cell is the immune cell according to any one of claims 3 to 17, 19 to 29, and 79 to 89. The combination according to claim 146 or 147, wherein the tyrosine kinase inhibitor is the tyrosine kinase inhibitor according to any one of claims 30 to 59.